# Patent

## Title
Methods of detecting sepsis

## Abstract

Methods of detecting sepsis in a sample from a patient are provided. Methods of detecting changes in expression of one or more microRNAs associated with sepsis are also provided. Compositions and kits are also provided.

## Background
This application claims priority to U.S. Provisional Application No. 61/149,277, filed Feb. 2, 2009, which is incorporated by reference herein in its entirety for any purpose.


1. BACKGROUND
Sepsis is the presence in the blood or other tissues of pathogenic microorganisms or their toxins combined with the host's inflammatory response, known as systemic inflammatory response syndrome (“SIRS”) caused by the infection. The immune response is mediated by a class of proteins called toll-like receptors (“TLR”) that recognize structurally-conserved molecules broadly shared by microorganisms but which are distinguishable from host molecules.
Once microorganisms have breached barriers such as the skin or intestinal tract, the body's TLRs recognize them and stimulate an immune response. Thus, in addition to symptoms caused by the microbial infection itself, sepsis is also characterized by symptoms of acute inflammation brought on by the host's immune response. These latter symptoms may include fever and elevated white blood cell count, or low white blood cell count and low body temperature. SIRS is characterized by hemodynamic compromise and resultant metabolic dysregulation, and may be accompanied by symptoms such as high heart rate, high respiratory rate and elevated body temperature. The immunological response also causes widespread activation of acute phase proteins, affecting the complement system and the coagulation pathways, which then cause damage to the vasculature and organs. Various neuroendocrine counter-regulatory systems are then activated as well, often compounding the problem.
Sepsis is often treated in the intensive care unit with intravenous fluids and antibiotics and/or antiviral compounds. However sepsis progresses quickly, and so even with immediate and aggressive treatment, severe sepsis can lead to organ failure and death. Severe sepsis is estimated to cause 215,000 deaths per year in the United States, more than acute myocardial infarction, stroke or pneumonia, which is likely due to late diagnosis or misdiagnosis of sepsis.
Thus, there is a need for early molecular markers in detecting sepsis.
2. SUMMARY
Methods for detecting the presence of sepsis in a subject are provided. In some embodiments, a method comprises detecting a level of at least one target RNA in a sample from the subject. In some embodiments, the at least one target RNA (1) is capable of specifically hybridizing to a nucleic acid having a sequence selected from SEQ ID NOs: 1 to 86; or (ii) comprises a sequence that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86; or (iii) comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, a method comprises comparing the level of the at least one target RNA in the sample to a normal level of the at least one target RNA. In some embodiments, a level of at least one target RNA in the sample that is greater than a normal level of the at least one target RNA indicates the presence of sepsis in the subject.
Methods for facilitating the detection of sepsis in a subject are also provided. In some embodiments, the method comprises detecting a level of at least one target RNA in a sample from the subject. In some embodiments, the at least one target RNA (i) is capable of specifically hybridizing to a nucleic acid having a sequence selected from SEQ ID NOs: 1 to 86; or (ii) comprises a sequence that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86; or (iii) comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, a method comprises communicating the results of the detection to a medical practitioner for the purpose of determining whether the subject has sepsis.
In some embodiments, detecting a level of at least one target RNA in a sample comprises hybridizing nucleic acids of the sample with at least one polynucleotide that is complementary to a target RNA in the sample or to a complement thereof. In some embodiments, a method further comprises detecting at least one complex comprising a polynucleotide hybridized to at least one nucleic acid selected from the target RNA, a DNA amplicon of the target RNA, and a complement of the target RNA.
In some embodiments, a method for detecting the presence of sepsis in a subject comprises obtaining a sample from the subject and providing the sample to a laboratory for detection of the level of at least one target RNA in the sample. In some embodiments, the at least one target RNA: (i) is capable of specifically hybridizing to a nucleic acid having a sequence selected from SEQ ID. NOs: 1 to 86; or (ii) comprises a sequence that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86; or (iii) comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, the method comprises receiving from the laboratory a communication indicating the level of at least one target RNA in the sample. In some embodiments, a level of at least one target RNA that is greater than a normal level of the at least one target RNA indicates the presence of sepsis.
In some embodiments, a method comprises detecting levels of at least two, at least three, at least five, or at least ten target RNAs. In some embodiments, detection of a level of at least one target RNA that is greater than a normal level of the at least one target RNA indicates the presence of sepsis. In some embodiments, detection of levels of at least two target RNAs that are greater than normal levels of the at least two target RNAs indicates the presence of sepsis. In some embodiments, detection of levels of at least three target RNAs that are greater than normal levels of the at least two target RNAs indicates the presence of sepsis. In some embodiments, detection of levels of at least five target RNAs that are greater than normal levels of the at least two target RNAs indicates the presence of sepsis.
In some embodiments, a method comprises detecting a level of at least one target RNA that (i) does not specifically hybridize to a nucleic acid having a sequence selected from SEQ ID NOs: 1 to 86; and (ii) does not comprise a sequence that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86; and (iii) does not comprise at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897.
In some embodiments, a synthetic polynucleotide is provided. In some embodiments, a synthetic polynucleotide comprises a first region, wherein the first region comprises a sequence of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 contiguous nucleotides that is identical or complementary to a sequence of at least 8 contiguous nucleotides of one of SEQ ID NOs: 1 to 67, 215 to 399, and 950. In some embodiments, the first region is identical or complementary to a region of a target RNA. In some embodiments, a synthetic polynucleotide comprises a second region that is not identical or complementary to a region of the target RNA. In some embodiments, a synthetic polynucleotide comprises a detectable label. In some embodiments, a synthetic polynucleotide comprises a FRET label.
In some embodiments, a composition is provided. In some embodiments, a composition comprises a plurality of synthetic polynucleotides. In some embodiments, a kit is provided. In some embodiments, a kit comprises a synthetic polynucleotide. In some embodiments, a kit comprises a composition. In some embodiments, a kit comprises at least one polymerase and/or dNTPs.
Further embodiments and details of the inventions are described below



3. BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows an electropherogram obtained on an Agilent Bioanalyser 2100 to assess the quality of total RNA purified as described in Example 1 from human monocyte cell line THP-1 after stimulation for 8 h with an agonist, Pam3CSK4.



4. DETAILED DESCRIPTION
4.1. Detecting Sepsis
4.1.1. General Methods
Methods detecting sepsis by measuring levels of microRNA species are provided. In some embodiments, elevated levels of microRNA species are indicative of sepsis. In some embodiments, reduced levels of microRNA species are indicative of sepsis. In some embodiments, the method comprises detecting an above-normal level of at least one target RNA that is capable of specifically hybridizing to a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, the method comprises detecting an above-normal level of at least one target RNA that comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of a sequence selected from SEQ ID NO.: 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, the method comprises detecting an above-normal level of at least one target RNA that comprises a sequence that is complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of a sequence selected from SEQ ID NO.:1 to 86. In some embodiments, the target RNA, in its mature form, comprises fewer than 30 nucleotides. The target RNA, in some embodiments, is a microRNA.
In the present disclosure, “a sequence selected from” encompasses both “one sequence selected from” and “one or more sequences selected from.” Thus, when “a sequence selected from” is used, it is to be understood that one, or more than one of the listed sequences may be chosen.
Detection of a level of target RNA that is greater than a normal level of target RNA indicates the presence of sepsis in the patient from whom the sample is taken sample. In some embodiments, the detecting is done quantitatively. In other embodiments, the detecting is done qualitatively. In some embodiments, detecting a target RNA comprises forming a complex comprising a polynucleotide and a nucleic acid selected from a target RNA, a DNA amplicon of a target RNA, and a complement of a target RNA. In some embodiments, the level of the complex is then detected and compared to a normal level of the same complex. The level of the complex, in some embodiments, correlates with the level of the target RNA in the sample.
“Sepsis” is an infection accompanied by an acute inflammatory reaction (systemic inflammatory response syndrome) with systemic manifestations associated with release of endogenous mediators of inflammation into the bloodstream. If left untreated, sepsis can become severe sepsis, which is often accompanied by the failure of at least one organ or septic shock, which is severe sepsis accompanied by organ hypoperfusion and hypotension that are poorly responsive to initial fluid resuscitation. The systemic inflammatory response is mediated by toll-like receptors (“TLRs”).
“Toll-like receptors” or “TLRs” are a class of proteins in vertebrates and invertebrates that recognize particular structurally conserved molecules on microorganisms that are distinguishable from host molecules, and which mediate immune cell responses. TLRs are located either on the surface of cells or in cellular compartments and are classified by the types of molecules they recognize and that stimulate them, as shown in Table 1.










TABLE 1





Receptor
Ligand(s)
Microorganism
TLR Location







TLR1
Multiple triacyl
Bacteria
Cell surface



lipopeptides


TLR2
Multiple glycolipids
Bacteria
Cell surface


(TLR2a,
Multiple lipopeptides
Bacteria


TLR2b)
Multiple lipoproteins
Bacteria



Lipoteichoic acid
Bacteria



Peptidoglycan
(Gram positive)




bacteria



Pam3CSK4
Bacteria


TLR3
Double-stranded
Viruses
Cell compartment



RNA, polyl:C


TLR4
Lipopolysaccharide
Gram-negative
Cell surface


(TLR4a,

bacteria


TLR4b)


TLR5
Flagellin
Bacteria
Cell surface


TLR7
Single-stranded RNA
Virus
Cell compartment


TLR9
Unmethylated CpG
Bacteria, viral
Cell compartment



DNA









Stimulation of various TLRs results in over-expression of one or more target RNAs, as shown in Table 2. In some embodiments, one or more target RNAs is over-expressed as a result of stimulation of a subset of TLRs that recognize bacteria (e.g., TLR1, TLR2, TLR4 or TLR5). In some embodiments, one or more target RNAs is over-expressed as a result of stimulation of a subset of TLRs that recognize viruses (e.g. TLR3 or TLR7). In some embodiments, one or more target RNAs is over-expressed as a result of stimulation of a subset of TLRs that recognize molecules common to both bacteria and viruses (TLR9). In some embodiments, one or more target RNAs is over-expressed as a result of stimulation of a subset of TLRs that recognize gram-negative bacteria (e.g., TLR4a and TLR4b). In some embodiments, one or more target RNAs is over-expressed as a result of stimulation of a subset of TLRs that recognize both gram-negative and gram-positive bacteria (e.g., TLR2a, TLR2b and TLR5). In some embodiments, one or more target RNAs is over-expressed as a result of stimulation of a subset of TLRs that recognize gram-positive bacteria, gram-negative bacteria and mycobacteria (e.g., TLR2a).
Table 2, below, lists 86 hybridization probes that have been found to be complimentary to, and to hybridize with, target RNAs in human monocytes stimulated with various toll-like receptor agonists (ligands). These target RNAs can be detected at elevated levels in stimulated THP-1 cells as demonstrated in Example 1. Sixty-seven of the probes are complementary to, and hybridize with, novel target RNA species that are expressed in human cells. The other nineteen probes are complementary to, and hybridize with, publicly known microRNAs that have been submitted by others to miRBase (microrna.sanger.ac.uk/; see Griffiths-Jones S. et al. (2007) Nucl. Acids Res. 36:154-158): hsa-miR-1227, hsa-miR-125b, hsa-miR-125 b, hsa-miR-142-3p, hsa-miR-155, hsa-miR-16, hsa-miR-195*, hsa-miR-214, hsa-miR-29b, hsa-miR-326, hsa-miR-371-3p, hsa-miR-371-5p, hsa-miR-374b*, hsa-miR-520c-5p, hsa-miR-526a, hsa-miR-518d-5p, hsa-miR-524-5p, hsa-miR-525-3p, hsa-miR-525-5p, hsa-miR-579, hsa-miR-885-3p and hsa-miR-99b). However, to the knowledge of the inventors, these known microRNAs have not been disclosed to have utility for detection of sepsis. The sequences of those microRNAs are shown in Table 4. Certain candidate microRNAs that may hybridize to certain probes listed in Table 2 are shown in Table 11.
Table 12, below, lists microRNAs that are present at elevated levels in a sepsis patient sample. Some pairs of microRNAs listed in Table 12 have the same sequences. In such instances, the precursor gene for that microRNA sequence is located at multiple locations in the genome, so the sequence may be from any of those genes. When a precursor gene for a particular microRNA sequence is present at multiple locations in the genome, multiple candidate names are shown (based on each of the precursor genes), with the same ranking and same sequence. One or more of those candidates may be upregulated in the sepsis patient sample. Some of the microRNAs listed in Table 12 are isomirs of one another. When multiple isomirs are listed in Table 12, one or more than one of the isomirs may be present at elevated levels in a sample from a patient with sepsis.
Table 14 lists microRNAs from miRBase that are present at elevated levels in a sepsis patient sample.
Table 16 lists microRNA star forms that are present at elevated levels in a sepsis patient sample. While the mature microRNAs for the listed star forms have been identified and are submitted into miRBase, none of the star forms in Table 16 have, to the inventors' knowledge, been previously identified or submitted to miRBase.
In some embodiments, a method comprises detecting multiple isomirs with a single probe. Detection of an elevated level of one or multiple isomirs is considered to be indicative of sepsis. When multiple microRNAs having the same sequence but are expressed from different genes, one or more of the genes may be upregulated in a sepsis patient. Detection of a microRNA expressed from any one of the genes is considered to be indicative of sepsis.
For convenience of reference herein, and not by way of limitation, some “target RNA” species are denominated “microRNAs” in the tables set forth herein and Example 1. In some embodiments, the target RNA is a single mature microRNA capable of specifically hybridizing to a hybridization probe set forth in Table 2. In some embodiments, a target RNA is a single mature microRNA that comprises a sequence that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NO.:1 to 86. In some embodiments, a target RNA is a single mature microRNA that comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, target RNA may include a plurality of target RNAs, all of which are capable of specifically hybridizing to a single complementary probe sequence (for example, when two or more target microRNAs are isomirs). In some embodiments, the so-denominated “microRNA” is one or more RNA species capable of specifically hybridizing to the respective hybridization probe, such that one or more target RNAs do not meet canonical definitions for mature microRNAs. In some embodiments, a target RNA is an mRNA. In some embodiments, the “target RNA” is a piwi-interacting RNA (piRNA), i.e., a small RNA expressed in animal cells that is distinct in size (26-31 nt) from microRNA and that forms distinct complexes with Piwi proteins that are involved in transcriptional gene silencing.
Mature human microRNAs are typically composed of 17-27 contiguous ribonucleotides, and often are 21 or 22 nucleotides in length. The sequences of some target microRNAs that can be detected in accordance with the present disclosure can be found within the pre-microRNA sequences shown in Tables 3, 13, 15, and 17 (SEQ ID NOs: 87 to 177, 948, 400 to 564, 949, 708 to 862, and 898 to 932). The sequences of some publicly known microRNAs are shown in Tables 4 and 14. Further, in some embodiments, a microRNA comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 contiguous nucleotides of a sequence in Table 11, 12, or 16 (SEQ ID NOs: 215 to 399, 950, and 863 to 897).
While not intending to be bound by theory, mammalian microRNAs mature as described herein. A gene coding for a microRNA is transcribed, leading to production of a microRNA precursor known as the “pri-microRNA” or “pri-miRNA.” The pri-miRNA can be part of a polycistronic RNA comprising multiple pri-miRNAs. In some circumstances, the pri-miRNA forms a hairpin with a stem and loop, which may comprise mismatched bases. The hairpin structure of the pri-miRNA is recognized by Drosha, which is an RNase III endonuclease protein. Drosha can recognize terminal loops in the pri-miRNA and cleave approximately two helical turns into the stem to produce a 60-70 nucleotide precursor known as the “pre-microRNA” or “pre-miRNA.” Drosha can cleave the pri-miRNA with a staggered cut typical of RNase III endonucleases yielding a pre-miRNA stem loop with a 5′ phosphate and an approximately 2-nucleotide 3′ overhang. Approximately one helical turn of the stem (about 10 nucleotides) extending beyond the Drosha cleavage site can be essential for efficient processing. The pre-miRNA is subsequently actively transported from the nucleus to the cytoplasm by Ran-GTP and the export receptor Exportin-5.
The pre-miRNA can be recognized by Dicer, another RNase III endonuclease. In some circumstances, Dicer recognizes the double-stranded stem of the pre-miRNA. Dicer may also recognize the 5′ phosphate and 3′ overhang at the base of the stem loop. Dicer may cleave off the terminal loop two helical turns away from the base of the stem loop leaving an additional 5′ phosphate and an approximately 2-nucleotide 3′ overhang. The resulting siRNA-like duplex, which may comprise mismatches, comprises the mature microRNA and a similar-sized fragment known as the microRNA*. The microRNA and microRNA* may be derived from opposing arms of the pri-miRNA and pre-miRNA. The mature microRNA is then loaded into the RNA-induced silencing complex (“RISC”), a ribonucleoprotein complex. In some cases, the microRNA* also has gene silencing or other activity.









TABLE 2









Bacterial Infection

















Gram+,








Probe
Myco-bacteria,
gram+,


gram+,



Probe Sequence
SEQ ID
Gram−
gram−
Gram−
Gram−
gram−


Probe
(5′ to 3′ without linker)
NO.
TLR2a
TLR2b
TLR4b
TLR4a
TLR5





10096-L3-1
TTCAGAATGTTAAGTCCCTATCCTTCGAT
1







 


10118-L4-1
AGAGCAACAGAGGGTTTGTGTAGGACTATGAGAGTGG
2








CG








 


10179-L4-1
TGGAACCTTTAGCAGCCAAACAGATTGCACAATCTC
3







 


10201-L4-1
CCCATTAAGAAATTGCAAGGCTAATAAAAATCATGC
4







 


10201-R4-1
GCAGGATTCTACCAGCCAGCCCTCAGGG
5







 


10225-L3-1
AATTAGGCAGTTAATATATTGTAACTAATA
6


11




 


10231-L3-1
GTGCAGCAGCCCGCGCCAGCCTCCGCAGCCGCC
7







 


10562_A-L4-1
CCCTACCTGTCAGTGTGACCATCACGAGCCTCCTGAG
8
17







ACCTCCTCTC








 


10630-L3-1
TTCATACAAACCTCCAACATTAAATTGCTA
9







 


11556-L4-1
ACAAACCACTAAGGAAGGAAGTGGCATACATTCTGCT
10







 


11744-R4-1
GGAAAATGTTTGGCGTCAAGTTCTTAAAAATGCTTCC
11
4.31


13




TCCTCTTTTTTCC








 


12381-L4-1
GACAGAGAGAGTGAGAGTGCGAGCTCACAGG
12
5.00






 


12428-R4-1
GCTCTTTAGCACTTTTACCCTTTGAAAATATAAAATC
13
7


5




AC








 


12432-L4-1
AAAGACTCATCAGATCCATTTCCAAAGTACAGCTC
14
15






 


12473-L4-1
TGCCAGAGGCTGTAATTGGTCTCAGGTAGTCTCTG
15
6


6



 


3817-L2-2
TCCCTGCAGCTGGCCAGCAATTACCGCCTGCCAG
16







 


3923-L3-1
AACACAAGAAGAAACGCCTGGTGCAGAGCC
17


11




 


3953-R3-2
ACTCCAGCCTCCGCCGCCTCAGCTTCCCGAGC
18







 


3995-L2-2
CTATAAAACTTCGAAAAGTCCCTCCTCCTCACGT
19



7



 


4214-R4-1
AAAGCTGGCAGGGCTTGTGAGCTCTGCAGG
20


12




 


4256-L1-1
AAATAATCAAGCTGTCACATCTTAAAAATC
21



14



 


4315_I-L4-1
ACACCTCTGCGCCCCTCAGGCGCCCTGGGCCTCGGCG
22








CCCCGCCCGTCCCAG
1.91







 


4440-L3-2
TTTGACATTCAGAGCACTGGGCAGAAATCACA
23

2





 


448-L4-1
GCTTCTGATGATCTATTAATGCAATACATCAGGGTGA
24








G








 


4483-L3-1
ATGTCATTGAAAGGGGGTATAATTGTGGTCTCC
25







 


4504-L3-1
ATTGATTTTACATTTTCCTGCCCTGCTAC
26







 


4972-R3-2
AGATCAAACAGGCTGTGAAGAAGCTCTATGAC
27


8




 


5230-L4-1
CCCAGGGTCACCCAACACACTCTGCCTTGAGCCTC
28







 


5316-R3-1
AAATTAAATTGACACTTTGAAGCATTAATCTA
29


56




 


5342-L3-1
CACCACCAAACCAAATGCCGCTGCTCTCCTTCCA
30




4.30


 


5491-L3-2
TGCCTCCGCGGCCGCAGGTAATGACCTGGAAGG
31







 


5598-R2-2
CTCCCACCTCCGTGAAGCTATTTTTAACTGTGCA
32







 


5619-R3-1
ATTGGATCAAACAGACGGGCACAATCA
33


35




 


5749-R3-1
ACAAGCATCATAATCCCCCTTTTGACTT
34
5.60

7
21



 


5956-L3-1
GCTCTGCCAACCCCAAATCCGTCAAGACGCATAG
35



5



 


6087-L4-1
CTGTCTCCATTACTGCCTGCCACCTTCTCCATC
36







 


6192-L3-1
AGATAAAAAACCACCCACCCAGCAC
37







 


6395-R1-2
GCTCAGAGCCAGTGTAATCCTCCTCTTGTGT
38



8



 


6428-R1-1
TGCCACTCTCATCACAGGGTGCAATAGCATA
39

14





 


6433-L4-1
CAGCTAGCTCTTGCTGCCTCTGCTGCTCAGCCTTCT
40







 


6511-R4-1
AGCTAACAATATATTAAAACATAATATCTATTTAGCA
41








GGT








 


6647-R2-1
CTCAGCCCCAGCTGGAGAATTTTTCCCCTCATTA
42







 


6693-L3-2
GAAAGAATTACACTTGACAGAGGCAGAGGAAAATGC
43







 


6998-L2-1
CAGCACAGTGGAGAAAAGTAACTGTCTTT
44







 


7094-L3-1
ATGACAGGATAATAACATTACATTAAAA
45


44




 


7158-R3-1
TACATTTATAGATTCCCTCTTCAGCCATA
46



8



 


7271-L2-1
CAGCTATTTTATTCTTGACATCAATTTCTGAAA
47


13




 


7411-L3-2
CTGCTGTTTTATTGTCACAGCTGGAGCCAGTTC
48


18




 


7824-R3-1
TGGTGCCAGCTTCATCGCCG
49



13



 


7828-R1-1
GCCAATCAATAATTCTGTGCCAAGCAACT
50







 


7997-L1-1
TCCGTTACACTAATTGCCATGATTTAGTCCAA
51







 


8012-L3-1
CCATCTTTACTAGATTTATAATTTGAG
52




39


 


8016-R3-1
AGAGGGGGTTGCTGAGCCGCCTGCAAGA
53







 


8075-L3-1
CCCAGCTACACCTCCACGCA
54







 


8433_D-L4-1
ACTAAAAGGAGCCGGAGGAGCTGGAGAGACGCGGGGC
55








CGAGCCGGG








 


8468-L3-2
AGCCATCCATTTAAATGAAAATCAGCACTGATACA
56

18

13
31


 


8505-L3-1
CTACGTGTGTCCTTTCTTTCACATTTGCTG
57

41





 


9229-R3-1
CTGACATTTATCTTCAGCATCTAG
58

16

43



 


9369-R3-2
GAATTTTCCCCTGCACAGTTAGGACAGGATGCATG
59
5






 


9391-R3-1
CAGCTGCCAGGGAGACATAGAAATTAAAAACAA
60




3


 


9576-L3-2
TCTGTTGATTGATTATATTTATCAGTGTAGAAGA
61







 


9638-R3-1
TGGTCATCAAACCTCAGCCTCTATCCCATCAA
62
6






 


9654-L4-1
GTTGAGAGTGAGCATAGCTTTGACTCTGCAAACTAAA
63
28







AGTTCCAGG








 


9688-L2-1
GCTAAATGGCCCCAGACTGTTCTGCTGCA
64
13






 


9840-L3-2
TTCAGGTTTTTATAAATCAGGATGTCAACAAAT
65




3


 


9850-L3-1
CTGCCAAGATACCTGATTT
66







 


9850-R3-1
CCAGAAGATGCAGAAGACAG
67

12


12


 


miR-1227
CTGGGGAAAAGGGTGGCACG
68







 


miR-125b
TCACAAGTTAGGGTCTCAGGGA
69



5



 


miR-142-3p
TCCATAAAGTAGGAAACACTACA
70







 


miR-155
ACCCCTATCACGATTAGCATTAA
71
1
2.50
1.42
1.33



 


miR-16
CGCCAATATTTACGTGCTGCTA
72







 


miR-195*
GGAGCAGCACAGCCAATATTGG
73

780
93
107



 


miR-214
ACTGCCTGTCTGTGCCTGCTGT
74
2352
1910





 


miR-29b
AACACTGATTTCAAATGGTGCTA
75







 


miR-326
CTGGAGGAAGGGCCCAGAGG
76


8
44
181


 


miR-371-3p
ACACTCAAAAGATGGCGGCACTT
77







 


miR-371-5p
AGTGCCCCCACAGTTTGAGT
78
4






 


miR-374b*
AATGATAATACAACCTGCTAAG
79

621





 


miR-520c-5p
CAGAAAGTGCTTCCCTCTAGAG
80







miR-526a









miR-518d-5p









miR-524-5p
GAGAAAGTGCTTCCCTTTGTAG
81







 


miR-525-3p
CGCTCTAAAGGGAAGCGCCTTC
82







 


miR-525-5p
AGAAAGTGCATCCCTCTGGAG
83







 


miR-579
AATCGCGGTTTATACCAAATGAA
84



1.29



 


miR-885-3p
TATCCACTACACCCCGCTGCCT
85







 


miR-99b
CGCAAGGTCGGTTCTACGGGTG
86
5
8

5















Bacterial and Viral
Viral infection













Infection
Viral
Viral




Unmethylated CpG
nucleic
nucleic




(bacterial, Viral)
acid
acid



Probe
TLR9
TLR3
TLR7






10096-L3-1

12.00




10118-L4-1






10179-L4-1

14.00




10201-L4-1

12.00




10201-R4-1

5.00




10225-L3-1






10231-L3-1


2.70



10562_A-L4-1






10630-L3-1
22.00





11556-L4-1

7.00




11744-R4-1






12381-L4-1






12428-R4-1






12432-L4-1






12473-L4-1






3817-L2-2


3.00



3923-L3-1






3953-R3-2
1.77





3995-L2-2






4214-R4-1






4256-L1-1






4315_I-L4-1






4440-L3-2






448-L4-1

7.00




4483-L3-1

14.00




4504-L3-1

8.00




4972-R3-2






5230-L4-1


7.00



5316-R3-1






5342-L3-1
2.70





5491-L3-2

7.00
12.00



5598-R2-2


2.30



5619-R3-1






5749-R3-1






5956-L3-1


11.00



6087-L4-1
5.00

1.00



6192-L3-1


8.00



6395-R1-2






6428-R1-1






6433-L4-1






6511-R4-1

5.00




6647-R2-1


1.70



6693-L3-2

4.60




6998-L2-1

9.00




7094-L3-1






7158-R3-1






7271-L2-1






7411-L3-2






7824-R3-1






7828-R1-1
73.00





7997-L1-1
238.00





8012-L3-1






8016-R3-1


13.00



8075-L3-1


5.00



8433_D-L4-1

7.40




8468-L3-2






8505-L3-1






9229-R3-1






9369-R3-2






9391-R3-1
5.00

3.00



9576-L3-2


10.00



9638-R3-1






9654-L4-1

14.00




9688-L2-1






9840-L3-2


3.00



9850-L3-1


11.00



9850-R3-1






miR-1227

7.00




miR-125b






miR-142-3p
1.87





miR-155

0.90




miR-16
2.39





miR-195*






miR-214






miR-29b
2.02





miR-326






miR-371-3p


11.00



miR-371-5p






miR-374b*






miR-520c-5p


9.00



miR-526a






miR-518d-5p






miR-524-5p


34.00



miR-525-3p


14.00



miR-525-5p


9.00



miR-579






miR-885-3p


3.80



miR-99b



















TABLE 3





Pre-


Pre-micro


microRNA
Chromosomal

RNA SEQ


Candidate
location
Pre-microRNA sequences
ID NO.


















10096-L3-1
5q14.3
AAGTCTTACAATTCAGGGATAGGAAGCTATGATTTACATAATGAACATGGCAAAGAGACCTATAAAG
87




AAATAAGACTT



 


10118-L4-1
9q33.3
CGCCACTCTCATAGTCCTACACAAACCCTCTGTTGCTCTTCTATTAGACCATGCTAACCAATTCTGA
88




GGGCTGTGAGGGGTAGGTG



 


10179-L4-1
3q13.31
GTTGAGATTGTGCAATCTGTTTGGCTGCTAAAGGTTCCAAATTATGTGGGCATTCTGCAGCCCCACA
89




GAGTGGTAGAATTTCTTC



 


10201-L4-1
6p12.2
GCATGATTTTTATTAGCCTTGCAATTTCTTAATGGGCTCCCCTGAGGGCTGGCTGGTAGAATCCTGC
90


 


10201-R4-1
6p12.2
GCATGATTTTTATTAGCCTTGCAATTTCTTAATGGGCTCCCCTGAGGGCTGGCTGGTAGAATCCTGC
91


 


10225-L3-1
10q26.13
TATTAGTTACAATATATTAACTGCCTAATTTAAAAATAAAACTATCTTTATGAAGGGCAATTAACCA
92




CTAAGTGTAATTGATA



 


10231-L3-1
9p11.2
GGCGGCTGCGGAGGCTGGCGCGGGCTGCTGCACCTTTAACGCTTTCTGGCGCTGACAGGCGGCGGCC
93




CAGCTAAAGTTCACAGCGCC



 


10562_A-L4-1
8p12
GAGAGGAGGTCTCAGGAGGCTCGTGATGGTCACACTGACAGGTAGGGCTTTCACTCCCATCCCCTCT
94




TGATACTCACCTGCCGCCCCCGACCCCTCTC



 


10630-L3-1
1p35.3
TAGCAATTTAATGTTGGAGGTTTGTATGAACTTGAAGCTTATTTCAGTTGGTTGCCTGGAACCTTCT
95




GCATTCTTTGCTG



 


11556-L4-1
10q23.32
AGCAGAATGTATGCCACTTCCTTCCTTAGTGGTTTGTCCGCCAACATTAACAGGCCATTGGGTGGAT
96




GAAGTAGGTAAATTTTGCT



 


11744-R4-1
1q32.2
GGAGAGCTGTGTTTCATGTGATTAGAGACTGTTTGTGCCTCTGTCCATTAGGAAAAAAGAGGAGGAA
97




GCATTTTTAAGAACTTGACGCCAAACATTTTCC



 


12381-L4-1
6p21.33
CCTGTGAGCTCGCACTCTCACTCTCTCTGTCTCTGTGTCAGGAGTGAATGGTGTGGGCTCCTCAGG
98


 


12428-R4-1
8p12
GCTAAAGTTGCTTCTGCCCTTTGAAAATATGAAAGCCCTTGAGTGATTTTATATTTTCAAAGGGTAA
99




AAGTGCTAAAGAGC



 


12432-L4-1
8p12
TGAGCTGTACTTTGGAAATGGATCTGATGAGTCTTTTAATAGAAGAAAATCATCATTATTTCCCAAG
100




AGCTCA



 


12473-L4-1
9p21.3
CAGAGACTACCTGAGACCAATTACAGCCTCTGGCATTTGTGCTGCTAAATTTGTAATGAGTTGCAGG
101




TGTTTGTG



 


3817-L2-2
2q13
TCTGGCAGGCGGTAATTGCTGGCCAGCTGCAGGGATTACAGCCCTGTGAGCTGTGTTCAGGGCCCTG
102




TGCCAGG



 


3923-L3-1
19p12
GGCTCTGCACCAGGCGTTTCTTCTTGTGTTTCCTCTTCTCTTCTGGAGAGGGATGAAGGAGATCCTT
103




TGTGAGAGGC



 


3953-R3-2
9q33.3
GCTCCTGCTCCGCCGCGGGAGCTGCTCCGGCGGCCGCAGGGCTCGCTCGGGAAGCTGAGGCGGCGGA
104




GGCTGGAGT



 


3995-L2-2
7p21.1
TGGCCTGACGTGAGGAGGAGGGACTTTTCGAAGTTTTATAGGAAAGTTTCCGCTTTCCAGTCCCCCT
105




CCCCCGTCCCA



 


4214-R4-1
8p12
GAGGTTGGACAGGCTTCTCCACACTGAGCTTTACAGGCCCGCTCCCTCCCCTGCAGAGCTCACAAGC
106




CCTGCCAGCTTT



 


4256-L1-1
2p22.1
GATTTTTAAGATGTGACAGCTTGATTATTTTACAAGGCCAAAACCCTGATTCAAGCCTGCAATTTTA
107




AGAATC



 


4315_I-L4-1
1q22
CTGGGACGGGCGGGGCGCCGAGGCCCAGGGCGCCTGAGGGGCGCAGAGGTGTCAGCGTGCAACCGCC
948




GCCCCCCAGCGTTCCCGCCACCACCGCCACCACCCTCAAAGCCCGG



 


4440-L3-2
7q11.22
GTGATGTGATTTCTGCCCAGTGCTCTGAATGTCAAACTGAAGAAATTCAGTGAAATGCGGGTAAACG
108




GCGGGAGTAACTATGAC



 


448-L4-1
8q21.12
CTCACCCTGATGTATTGCATTAATAGATCATCAGAAGCAGTTGTCATTCCAGTGATATATTAGTGCA
109




ATACATGAGAATGAG



 


4483-L3-1
16q12.1
GGAGACCACAATTATACCCCCTTTCAATGACATGTCTGGGGTTGCAGTGACTCCAGACAAAGAAGCT
110




GAAATGTATGAAAGTTTCC



 


4504-L3-1
13q14.3
GTAGCAGGGCAGGAAAATGTAAAATCAATAAATAATCAGGCTGAATTTTAATTGAATATATTCCTAA
111




GGCCATGCTGAC



 


4972-R3-2
3p26.3
CATCAGGTCGAATCAGGGTGTTGACCTTGGCCACATCAATGTCATAGAGCTTCTTCACAGCCTGTTT
112




GATCTGGTG



 


5230-L4-1
9q33.2
GAGGCTCAAGGCAGAGTGTGTTGGGTGACCCTGGGTAGGGCTTGGTTGGCCACTTACCACATGGTTG
113




CCACTGGGGCCTT



 


5316-R3-1
13q14.3
CTGAACAAATAGCAGATGTTGATGAATATTAATTTGTGCTTAGATTAATGCTTCAAAGTGTCAATTT
114




AATTTCTGTTTTACTATTCAG



 


5342-L3-1
8p21.2
TGGAAGGAGAGCAGCGGCATTTGGTTTGGTGGTGGGCAGATTTTCTTTTACGACTGCTAAATGCCTG
115




CCTTTCTCCCCA



 


5491-L3-2
6p21.33
GCCTCCCCTTCCAGGTCATTACCTGCGGCCGCGGAGGCAACAGCTGCCACCATGGCCTGATGAGTGA
116




TCTGGTGGGCGACGGC



 


5598-R2-2
11q13.2
CTCCCACGGCCTGAAGCTGCTGCCAAGCTATTTTTGGTTCTGCACAGTTAAAAATAGCTTCACGGAG
117




GTGGGAG



 


5619-R3-1
15q25.3
CTCATTGAGGGAAGATTGAGCAGAACTGGCATTGCTTGCTTTCGTCAAATTGATTGTGCCCGTCTGT
118




TTGATCCAATTCAGTGAG



 


5749-R3-1
18q23
ACAGGCTCATCCCTCTGAACAGATGAGATTAGTCGATCATGTAAAGTCAAAAGGGGGATTATGATGC
119




TTGT



 


5956-L3-1
1q41
CTATGCGTCTTGACGGATTTGGGGTTGGCAGAGCAGGCTGCCCCTGCTTTCTATCCCCATTCAGTCC
120




ACTTATAG



 


6087-L4-1
9q33.1
GATGGAGAAGGTGGCAGGCAGTAATGGAGACAGAATTTCTGTTAACTGCTGTAATTAATGTTATGTC
121




TCATC



 


6192-L3-1
11q25
GTGCTGGGTGGGTGGTTTTTTATCTTCACGGATTTATGGAGTCCTTAAAACATCTGTTCCGTTCTGA
122




TTCCCCCGCTCAGTAC



 


6395-R1-2
14q11.2
GTTCCGAGGCAGGCTTTCCTCCTCTCTGCAGGGGAGAGGCTCCCTCACACAAGAGGAGGATTACACT
123




GGCTCTGAGC



 


6428-R1-1
2p24.1
AAACTGGAACACATTCCTCAAGGGAGCAGGAAAGCATGAGAAGACAGTATGCTATTGCACCCTGTGA
124




TGAGAGTGGCAGTTT



 


6433-L4-1
1q24.2
AGAAGGCTGAGCAGCAGAGGCAGCAAGAGCTAGCTGCACATACCCAGCAACAGCCTTCCACTTCTGA
125




TCAGTCTTCT



 


6511-R4-1
1q24.1
AGCAAATTTACTATTGGGAATAAATATTTGATGCAGGTGAACACCTGCTAAATAGATATTATGTTTT
126




AATATATTGTTAGCT



 


6647-R2-1
1q23.3
CTCAGTATCTTCAGCTTGGGAAACTGACCTCGTTAATTTTAATGAGGGGAAAAATTCTCCAGCTGGG
127




GCTGAG



 


6693-L3-2
11q24.1
GACTGCAGGCATTTTCCTCTGCCTCTGTCAAGTGTAATTCTTTCTTGATGAATGACAAGGCAGGATA
128




ATAGGCTGTGGTC



 


6998-L2-1
11p14.1
AAAGACAGTTACTTTTCTCCACTGTGCTGCTACCACCAATTTGGTGGCTATTAATAGCTGGCAGATT
129




AACTTCTTT



 


7094-L3-1
2p23.2
TTTTAATGTAATGTTATTATCCTGTCATTTAATTTAGCAATGACAAGTGATGATGAGATTTTGATTT
130




GCATTAGAA



 


7158-R3-1
3q13.31
ATTCAACACAGATTCAGGTGCTCTCAACAGCCATGAAAATATATGGCTGAAGAGGGAATCTATAAAT
131




GTAATGAAT



 


7271-L2-1
11p12
AGGTGATGTTTTCAGAAATTGATGTCAAGAATAAAATAGCTGTTGGCAGTTACAACTGTTTGGATGT
132




CATTTTACAAAACAATTGCCT



 


7411-L3-2
18q22.3
GAGTGTGAACTGGCTCCAGCTGTGACAATAAAACAGCAGGTGGCTGCTGTCATTAGGGGTGGCAGAT
133




GAGGCAGGGGACTAACATTC



 


7824-R3-1
6q16.2
CCTGGATGCTGTTTCATTATGTAGAGTCAGGCAAAAGACAGACGGATGTGTGTGTGAGGCGGCGATG
134




AAGCTGGCACCAGG



 


7828-R1-1
11q24.3
GAGATTAGCGAAAGGGATTCTGGCCAAATCCCTGATCAAGTTGCTTGGCACAGAATTATTGATTGGC
135




AAATCTT



 


7997-L1-1
2q31.1
TTGGACTAAATCATGGCAATTAGTGTAACGGAAATGTTTACAGCAATCTCTGATGGCAGTTCTACTA
136




ATGCAATGATTTAGCTCAA



 


8012-L3-1
3q11.2
AAATATTTATGTACTCAAATTATAAATCTAGTAAAGATGGCATTTCACCTTATACTAGTTATTTATT
137




AATAATGAGAGCTGTATTT



 


8016-R3-1
12q21.1
AGAGGGGTGACTGCGGGGCTTGTTGCGCTGAAGATTTACAATGTACTTCTTGCAGGCGGCTCAGCAA
138




CCCCCTCT



 


8075-L3-1
10q22.1
CAGCTGGCCTGGTGCCCTGGTGCGTGGAGGTGTAGCTGGGCTCTGACCCAGCTCCTCAAACAGGTTC
139




CATATGGCCCTCCCGGCTG



 


8433_D-L4-1
17q25.3
CCCGGCTCGGCCCCGCGTCTCTCCAGCTCCTCCGGCTCCTTTTAGTGCATAAATTAGTGATGGCATT
140




TCCCGGAGAGCGGAGCACAACACAGGGCGCCGGGCTCGGG



 


8468-L3-2
15q23
GAGAGATTGTATCAGTGCTGATTTTCATTTAAATGGATGGCTATGAGATTTTTTAAAGCATGCCAAA
141




AATCTGTTTGTACATCTCTC



 


8505-L3-1
2p16.1
CAGCAAATGTGAAAGAAAGGACACACGTAGGTACTGTCATTTAGGTAATGTCATCTATGATCAGTTT
142




TTGTTTCATTTTTTGCTG



 


9229-R3-1
11q25
CTGACATTTACTTTCACTCATGAGCACAGGGGTGACCAGCCCCACCAGTCCTAGATGCTGAAGATAA
143




ATGTCAG



 


9369-R3-2
Xq26.3
TCCCCTGATTTCCCTCTGTGGAAGAATGTGTGAATTCACATGCATCCTGTCCTAACTGTGCAGGGGA
144




AAATTCCAGTCAGGGGA



 


9391-R3-1
2p14
TAGCTGCCTCAGAGTAGAAAATAAAACTCAACAAGATTTTATCTTGTTTTTAATTTCTATGTCTCCC
145




TGGCAGCTG



 


9576-L3-2
1p21.2
TTAAATTCTTCTACACTGATAAATATAATCAATCAACAGAGAACATGCTCTGAGGAATTAATTGTTG
146




TCAGTTGATGTATTTAA



 


9638-R3-1
8p21.3
GCTATAATGGAAAACTGATGGCTTTTATCTCCCCAACTTTATGACTATTGATGGGATAGAGGCTGAG
147




GTTTGATGACCATTTAATAGC



 


9654-L4-1
3p25.3
CCTGGAACTTTTAGTTTGCAGAGTCAAAGCTATGCTCACTCTCAACAATTGTAGAGAGGCTTTCTGG
148




CTGGGCAATCTAAAAAACCGGG



 


9688-L2-1
Xq26.2
TGCAGCAGAACAGTCTGGGGCCATTTAGCTTAGGGGCAAATAGTTCCTCATACTTCAAAGAGCCCTA
149




AGGACATTGCTGCA



 


9840-L3-2
5q14.1
CTTGTATTTGTTGACATCCTGATTTATAAAAACCTGAACAAGTTCAGTTTCAATAATTCTTTTTGTT
150




CAAGGAACACAAG



 


9850-L3-1
5p15.2
CCAGGAGAAATCAGGTATCTTGGCAGTGTGACCACCATGAATAAACAACAACTCTGGTGGCCTGTCT
151




TCTGCATCTTCTGG



 


9850-R3-1
5p15.2
CCAGGAGAAATCAGGTATCTTGGCAGTGTGACCACCATGAATAAACAACAACTCTGGTGGCCTGTCT
152




TCTGCATCTTCTGG



 


miR-1227
19p13.3
GTGGGGCCAGGCGGTGGTGGGCACTGCTGGGGTGGGCACAGCAGCCATGCAGAGCGGGCATTTGACC
153




CCGTGCCACCCTTTTCCCCAG



 


miR-125b
21q21.1
ACCAGACTTTTCCTAGTCCCTGAGACCCTAACTTGTGAGGTATTTTAGTAACATCACAAGTCAGGCT
154




CTTGGGACCTAGGCGGAGGGGA



 


miR-125b
11q24.1
TGCGCTCCTCTCAGTCCCTGAGACCCTAACTTGTGATGTTTACCGTTTAAATCCACGGGTTAGGCTC
155




TTGGGAGCTGCGAGTCGTGCT



 


miR-142-3p
17q23.2
GACAGTGCAGTCACCCATAAAGTAGAAAGCACTACTAACAGCACTGGAGGGTGTAGTGTTTCCTACT
156




TTATGGATGAGTGTACTGTG



 


miR-155
21q21.3
CTGTTAATGCTAATCGTGATAGGGGTTTTTGCCTCCAACTGACTCCTACATATTAGCATTAACAG
157


 


miR-16
13q14.2
GTCAGCAGTGCCTTAGCAGCACGTAAATATTGGCGTTAAGATTCTAAAATTATCTCCAGTATTAACT
158




GTGCTGCTGAAGTAAGGTTGAC



 


miR-16
3q25.33
GTTCCACTCTAGCAGCACGTAAATATTGGCGTAGTGAAATATATATTAAACACCAATATTACTGTGC
159




TGCTTTAGTGTGAC



 


miR-195*
17p13.1
AGCTTCCCTGGCTCTAGCAGCACAGAAATATTGGCACAGGGAAGCGAGTCTGCCAATATTGGCTGTG
160




CTGCTCCAGGCAGGGTGGTG



 


miR-214
1q24.3
GGCCTGGCTGGACAGAGTTGTCATGTGTCTGCCTGTCTACACTTGCTGTGCAGAACATCCGCTCACC
161




TGTACAGCAGGCACAGACAGGCAGTCACATGACAACCCAGCCT



 


miR-29b
7q32.3
CTTCAGGAAGCTGGTTTCATATGGTGGTTTAGATTTAAATAGTGATTGTCTAGCACCATTTGAAATC
162




AGTGTTCTTGGGGG



 


miR-29b
1q32.2
CTTCTGGAAGCTGGTTTCACATGGTGGCTTAGATTTTTCCATCTTTGTATCTAGCACCATTTGAAAT
163




CAGTGTTTTAGGAG



 


miR-326
11q13.4
CTCATCTGTCTGTTGGGCTGGAGGCAGGGCCTTTGTGAAGGCGGGTGGTGCTCAGATCGCCTCTGGG
164




CCCTTCCTCCAGCCCCGAGGCGGATTCA



 


miR-371-3p
19q13.42
GTGGCACTCAAACTGTGGGGGCACTTTCTGCTCTCTGGTGAAAGTGCCGCCATCTTTTGAGTGTTAC
165


 


miR-371-5p
19q13.42
GTGGCACTCAAACTGTGGGGGCACTTTCTGCTCTCTGGTGAAAGTGCCGCCATCTTTTGAGTGTTAC
166


 


miR-374b*
Xq13.2
ACTCGGATGGATATAATACAACCTGCTAAGTGTCCTAGCACTTAGCAGGTTGTATTATCATTGTCCG
167




TGTCT



 


miR-518d-5p
19q13.42
TCCCATGCTGTGACCCTCTAGAGGGAAGCACTTTCTGTTGTCTGAAAGAAACCAAAGCGCTTCCCTT
168




TGGAGCGTTACGGTTTGAGA



 


miR-520c-5p
19q13.42
TCTCAGGCTGTCGTCCTCTAGAGGGAAGCACTTTCTGTTGTCTGAAAGAAAAGAAAGTGCTTCCTTT
169




TAGAGGGTTACCGTTTGAGA



 


miR-526a
19q13.42
GTGACCCTCTAGAGGGAAGCACTTTCTGTTGAAAGAAAAGAACATGCATCCTTTCAGAGGGTTAC
170


 


miR-526a
19q13.42
CTCAGGCTGTGACCCTCTAGAGGGAAGCACTTTCTGTTGCTTGAAAGAAGAGAAAGCGCTTCCTTTT
171




AGAGGATTACTCTTTGAG



 


miR-524-5p
19q13.42
TCTCATGCTGTGACCCTACAAAGGGAAGCACTTTCTCTTGTCCAAAGGAAAAGAAGGCGCTTCCCTT
172




TGGAGTGTTACGGTTTGAGA



 


miR-525-3p
19q13.42
CTCAAGCTGTGACTCTCCAGAGGGATGCACTTTCTCTTATGTGAAAAAAAAGAAGGCGCTTCCCTTT
173




AGAGCGTTACGGTTTGGG



 


miR-525-5p
19q.13.42
CTCAAGCTGTGACTCTCCAGAGGGATGCACTTTCTCTTATGTGAAAAAAAAGAAGGCGCTTCCCTTT
174




AGAGCGTTACGGTTTGGG



 


miR-579
5p13.3
CATATTAGGTTAATGCAAAAGTAATCGCGGTTTGTGCCAGATGACGATTTGAATTAATAAATTCATT
175




TGGTATAAACCGCGATTATTTTTGCATCAAC



 


miR-885-3p
3p25.3
CCGCACTCTCTCCATTACACTACCCTGCCTCTTCTCCATGAGAGGCAGCGGGGTGTAGTGGATAGAG
176




CACGGGT



 


miR-99b
19q13.41
GGCACCCACCCGTAGAACCGACCTTGCGGGGCCTTCGCCGCACACAAGCTCGTGTCTGTGGGTCCGT
177




GTC
















TABLE 4







Mature microRNA Sequences (5′ to 3′)









microRNA
sequence
SEQ ID NO





miR-1227
CGUGCCACCCUUUUCCCCAG
196


 


miR-125b
UCCCUGAGACCCUAACUUGUGA
197


 


miR-142-3p
CAUAAAGUAGAAAGCACUACU
198


 


miR-155
UUAAUGCUAAUCGUGAUAGGGGU
199


 


miR-16
UAGCAGCACGUAAAUAUUGGCG
200


 


miR-195*
CCAAUAUUGGCUGUGCUGCUCC
201


 


miR-214
ACAGCAGGCACAGACAGGCAGU
202


 


miR-29b
UAGCACCAUUUGAAAUCAGUGUU
203


 


miR-326
CCUCUGGGCCCUUCCUCCAG
204


 


miR-371-3p
AAGUGCCGCCAUCUUUUGAGUGU
205


 


miR-371-5p
ACUCAAACUGUGGGGGCACU
206


 


miR-374b*
CUUAGCAGGUUGUAUUAUCAUU
207


 


miR-518d-5p
CUCUAGAGGGAAGCACUUUCUG
208


miR-520c-5p




miR-526a




 


miR-524-5p
CUACAAAGGGAAGCACUUUCUC
209


 


miR-525-3p
GAAGGCGCUUCCCUUUAGAGCG
210


 


miR-525-5p
CUCCAGAGGGAUGCACUUUCU
211


 


miR-579
UUCAUUUGGUAUAAACCGCGAUU
212


 


miR-885-3p
AGGCAGCGGGGUGUAGUGGAUA
213


 


miR-99b
CACCCGUAGAACCGACCUUGCG
214









In Table 2, the expression levels of target RNAs measured for stimulation of each of the identified TLRs are expressed as fold-changes in expression relative to expression levels measured in total RNA from human monocytes of healthy donors (see Example 1).
In some embodiments, target RNAs can be measured in samples collected at one or more times from a patient to monitor the status or progress of sepsis in the patient.
In some embodiments, the clinical sample to be tested is obtained from individuals who exhibit one or more symptoms of a systemic inflammatory response, including a body temperature greater than 38° C. or less than 36° C., a heart rate greater than 90 beats/minute, a respiratory rate greater than 20 breaths/min (or Paco2 less than 32 mm Hg), and a white blood cell count greater than 12,000 cells/μL or less than 4000 cells/μL, or with a content of greater than 10% immature forms. In some embodiments, the clinical sample to be tested is obtained from individuals who exhibit two or more of the above-described symptoms. In some embodiments, the clinical sample to be tested is obtained from asymptomatic individuals who are at risk for contracting sepsis, such as individuals who are elderly, immuno-compromised, critically ill, or are currently patients in, or have recently been discharged from, a hospital.
In some embodiments, the methods described herein are used for early detection of sepsis in a sample of human cells, such as those obtained by routine blood test. In some embodiments, the sample of human cells is a sample of human leukocytes. In some embodiments, the sample of human cells is a sample of human monocytes. Although for simplicity the discussion below refers to a sample of human monocytes, the skilled artisan will appreciate that the sample of human cells that can be used in the disclosed methods can include any human cells in which TLRs are expressed.
Thus, in some embodiments, methods of the present disclosure can be used for routine screening of individuals at risk for sepsis. In some embodiments, methods herein are used to (1) screen individuals who are elderly, (2) screen individuals who are immuno-compromised, (3) screen individuals who are critically ill or (4) screen individuals who are patients in, or have recently been discharged from, a hospital. In some embodiments, methods herein are used to screen neonates (less than 90 days old) with fever.
In some embodiments, the methods described herein can be used to determine the source of the underlying infection in a septic individual for targeted treatment of the underlying infection. In some embodiments, an increase in expression levels of one or more target RNAs associated with the stimulation of TLR2a, TLR2b, TLR4a, TLR4b or TLR5 indicates the presence of a bacterial infection in a septic individual. In some embodiments, an increase in expression levels of one or more target RNAs associated with the stimulation of TLR4a or TLR4b indicates the presence of an infection of gram-negative bacterial infection in the septic individual. In some embodiments, an increase in expression levels of one or more target RNAs associated with stimulation of TLR2a. TLR2b or TLR5 without concomitant stimulation of either TLR4a or TLR4b indicates the presence of a gram-positive bacterial infection in the septic individual. In some embodiments, an increase in expression levels of one or more target RNAs associated with stimulation of TLR2a without concomitant stimulation of TLR4a or TLR4b indicates the presence of either a gram-positive bacterial infection or a mycobacterial infection. In some embodiments, an increase in expression levels of one or more target RNAs associated with the stimulation of TLR3 or TLR7 indicates the presence of a viral infection. In some embodiments, an increase in expression levels of one or more target RNAs associated with the stimulation of TLR9 indicates the presence of a viral infection and/or a bacterial infection. In some embodiments, an increase in expression levels of: (i) one or more target RNAs associated with stimulation of TLR2a, TLR2b, TLR4a, TLR4b or TLR5; and (ii) one or more target RNAs associated with stimulation of TLR3 or TLR7 indicates the presence of both viral and bacterial infection.
In some embodiments, the methods described herein can be used to assess the effectiveness of a treatment for sepsis in a patient. In some embodiments, the target RNA expression levels are determined at various times during the treatment, and are compared to target RNA expression levels from an archival sample taken from the patient, e.g., by blood test, before the manifestation of any signs of sepsis or before beginning treatment. Ideally, target RNA expression levels in the normal blood sample evidence no aberrant changes in target RNA expression levels. Thus, in such embodiments, the progress of treatment of an individual with sepsis can be assessed by comparison to a sample from the same individual when he was healthy or prior to beginning treatment.
In some embodiments, the sample to be tested is a bodily fluid, such as blood, sputum, mucus, saliva, urine, semen, etc. In some embodiments, a sample to be tested is a blood sample. In some embodiments, the blood sample is whole blood, plasma, serum, or blood cells. In some embodiments, the blood sample is separated monocytes and/or lymphocytes. Monocytes and/or lymphocytes can be separated from whole blood by any method. In some embodiments, monocytes can be separated from whole blood or a fractionated or separated portion of whole blood using antibodies, e.g., to a cell surface receptor on the monocytes (such as CD14). In some such embodiments, the antibodies are coupled to beads, such as magnetic beads.
The clinical sample to be tested is, in some embodiments, freshly obtained. In other embodiments, the sample is a fresh frozen specimen.
In embodiments in which the method comprises detecting expression of more than one target RNA, the expression levels of the plurality of target RNAs may be detected concurrently or simultaneously in the same assay reaction. In some embodiments, expression levels are detected concurrently or simultaneously in separate assay reactions. In some embodiments, expression levels are detected at different times, e.g., in serial assay reactions.
In some embodiments, a method comprises detecting the level of at least one target RNA in a sample from a subject, wherein detection of a level of at least one target RNA that is greater than a normal level of the at least one target RNA indicates the presence of sepsis in the subject. In some embodiments, a method comprises detecting the level of at least one target RNA in a sample from a subject and comparing the level of the at least one target RNA in the sample to a normal level of the at least one target RNA, wherein a level of at least one target RNA in the sample that is greater than a normal level of the at least one target RNA indicates the presence of sepsis in the subject.
In some embodiments, a method of facilitating diagnosis of sepsis in a subject is provided. Such methods comprise detecting the level of at least one target RNA in a sample from the subject. In some embodiments, information concerning the level of at least one target RNA in the sample from the subject is communicated to a medical practitioner. A “medical practitioner,” as used herein, refers to an individual or entity that diagnoses and/or treats patients, such as a hospital, a clinic, a physician's office, a physician, a nurse, or an agent of any of the aforementioned entities and individuals. In some embodiments, detecting the level of at least one target RNA is carried out at a laboratory that has received the subject's sample from the medical practitioner or agent of the medical practitioner. The laboratory carries out the detection by any method, including those described herein, and then communicates the results to the medical practitioner. A result is “communicated,” as used herein, when it is provided by any means to the medical practitioner. In some embodiments, such communication may be oral or written, may be by telephone, in person, by e-mail, by mail or other courier, or may be made by directly depositing the information into, e.g., a database accessible by the medical practitioner, including databases not controlled by the medical practitioner. In some embodiments, the information is maintained in electronic form. In some embodiments, the information can be stored in a memory or other computer readable medium, such as RAM, ROM, EEPROM, flash memory, computer chips, digital video discs (DVD), compact discs (CDs), hard disk drives (HDD), magnetic tape, etc.
In some embodiments, methods of detecting the presence sepsis are provided. In some embodiments, methods of diagnosing sepsis are provided. In some embodiments, the method comprises obtaining a sample from a subject and providing the sample to a laboratory for detection of at least one target RNA level in the sample. In some embodiments, the method further comprises receiving a communication from the laboratory that indicates the at least one target RNA level in the sample. In some embodiments, sepsis is present if the level of at least one target RNA in the sample is greater than a normal level of the at least one target RNA. A “laboratory,” as used herein, is any facility that detects the level of at least one target RNA in a sample by any method, including the methods described herein, and communicates the level to a medical practitioner. In some embodiments, a laboratory is under the control of a medical practitioner. In some embodiments, a laboratory is not under the control of the medical practitioner.
When a laboratory communicates the level of at least one target RNA to a medical practitioner, in some embodiments, the laboratory communicates a numerical value representing the level of at least one target RNA in the sample, with or without providing a numerical value for a normal level. In some embodiments, the laboratory communicates the level of at least one target RNA by providing a qualitative value, such as “high,” “elevated.” etc.
As used herein, when a method relates to detecting sepsis, determining the presence of sepsis, and/or diagnosing sepsis, the method includes activities in which the steps of the method are carried out, but the result is negative for the presence of sepsis. That is, detecting, determining, and diagnosing sepsis include instances of carrying out the methods that result in either positive or negative results (e.g., whether target RNA levels are normal or greater than normal).
As used herein, the term “subject” means a human. In some embodiments, the methods described herein may be used on samples from non-human animals.
The common, or coordinate, expression of target RNAs that are physically proximal to one another in the genome permits the informative use of such chromosome-proximal target RNAs in methods herein.
Table 3 identifies the chromosomal location of each of the 86 target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1 to 86 in Table 2. Table 13 identifies the chromosomal location of the target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 226 to 399 and 950 in Table 12. Table 15 identifies the chromosomal location of the target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 565 to 707 in Table 14. Table 17 identifies the chromosomal location of the target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 863 to 897 in Table 16. Thus, in some embodiments, the level of expression of one or more target RNAs located within about 1 kilobase (kb), within about 2 kb, within about 5 kb, within about 10 kb, within about 20 kb, within about 30 kb, within about 40 kb, and even within about 50 kb of the chromosomal locations in Table 2 and Table 14 is detected in lieu of, or in addition to, measurement of expression of the respective tabulated target RNA in the methods described herein. See Baskerville, S. and Bartel D. P. (2005) RNA 11:241-247.
In some embodiments, in combination with detecting one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs:1 to 67 and/or detecting one or more target RNAs comprising at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897 and/or detecting one or more target RNAs that comprise a sequence that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs:1 to 67, methods herein further comprise detecting the level(s) of expression of at least one microRNA from the human miRNome.
In some embodiments, at least one target RNA is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, at least one target RNA comprises at least 15 contiguous nucleotides that are complementary to at least a portion of a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, at least one target RNA comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, a target RNA, in its mature form, comprises fewer than 30 nucleotides. In some embodiments, a target RNA is a microRNA.
In some embodiments, more than one target RNA is detected simultaneously in a single reaction. In some embodiments, at least 2, at least 3, at least 5, or at least 10 target RNAs are detected simultaneously in a single reaction. In some embodiments, all target RNAs are detected simultaneously in a single reaction.
In some embodiments, an increase in expression of one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NO: 1 to 86 in Table 2 in a sample is indicative of the presence of sepsis in an individual from whom the sample of blood or tissue has been taken. In some embodiments, an increase in expression of one or more target RNAs that comprise at least 15 contiguous nucleotides that are complementary to at least a portion of a sequence selected from SEQ ID NO: 1 to 86 in Table 2 in a sample is indicative of the presence of sepsis in an individual from whom the sample of blood or tissue has been taken. In some embodiments, an increase in expression of one or more target RNAs that comprise at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897 in a sample is indicative of the presence of sepsis in an individual from whom the sample of blood or tissue has been taken.
In some embodiments, an increase in expression of one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 35, 36, 37, 41, 42, 43, 44, 53, 54, 55, 60, 61, 63, 65, 66, 68, 71, 77, 80, 81, 82, 83 or 85 in Table 2 is indicative of the presence of sepsis caused by viral infection.
In some embodiments, an increase in expression of one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NO: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 or 86 in Table 2 in a sample of human monocytes is indicative of the presence of sepsis caused by a bacterial infection. In some embodiments, an increase in expression of one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NO: 6, 11, 13, 15, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 56, 58, 69, 71, 73, 76, 84 or 86 in Table 2 in a sample of human monocytes is indicative of the presence of sepsis caused by an infection of gram-negative bacteria. In some embodiments, an increase in expression of one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NO: 23, 30, 39, 52, 57, 60 65, 67 or 79 in Table 2 in a sample of human monocytes is indicative of the presence of sepsis caused by an infection of gram positive bacteria. In some embodiments, an increase in expression of one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NO: 8, 14, 59, 62, 63, 64, 74 or 78 in Table 2 in a sample of human monocytes is indicative of the presence of sepsis caused by an infection of gram positive bacteria or mycobacteria.
In some embodiments, an increase in expression of one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NO: SEQ ID NO: 9, 50, 51, 70, 72 or 75 in Table 2 in a sample of human monocytes is indicative of the presence of unmethylated CpG nucleic acids caused by a bacterial and/or a viral infection.
In some embodiments, an increase in expression of one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NO: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 35, 36, 37, 41, 42, 43, 44, 53, 54, 55, 60, 61, 63, 65, 66, 68, 71, 77, 80, 81, 82, 83 or 85 in Table 2 in a sample of human monocytes is indicative of stimulation of a toll-like receptor that recognizes virally-derived molecules. In some embodiments, these toll-like receptors are selected from TLR3 and TLR7.
In some embodiments, an increase in expression of one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NO: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 or 86 in Table 2 in a sample of human monocytes is indicative of stimulation of a toll-like receptor that recognizes bacterially-derived molecules. In some embodiments, these toll-like receptors are selected from TLR2a, TLR2b, TLR4a, TLR4b and TLR5. In some embodiments, an increase in expression of one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NO: 6, 11, 13, 15, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 56, 58, 69, 71, 73, 76, 84 or 86 in Table 2 in a sample of human monocytes is indicative of stimulation of a toll-like receptor that recognizes molecules derived from gram-negative bacteria. In some embodiments, these toll-like receptors are selected from TLR2a, TLR2b, TLR4a, TLR4b and TLR5. In some embodiments, an increase in expression of one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NO: 23, 30, 39, 52, 57, 60 65, 67 or 79 in Table 2 in a sample of human monocytes is indicative of stimulation of a toll-like receptor that recognizes molecules derived from gram-positive bacteria. In some embodiments, these toll-like receptors are selected from TLR2a, TLR2b and TLR5. In some embodiments, an increase in expression of one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NO: 8, 14, 59, 62, 63, 64, 74 or 78 in Table 2 in a sample of human monocytes is indicative of stimulation of a toll-like receptor that recognizes molecules derived from gram-positive bacteria or mycobacteria, such as TLR2a.
In some embodiments, an increase in expression of one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NO: 9, 50, 51, 70, 72 or 75 in Table 2 in a sample of human monocytes is indicative of stimulation of TLR9, which recognizes unmethylated CpG nucleic acids caused by a bacterial and/or a viral infection.
In some embodiments, an increase in expression of one or more target RNAs comprising at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 226 to 289, 565 to 604, and 863 to 868 in a sample of human monocytes is indicative of sepsis. In some embodiments, an increase in expression of one or more target RNAs comprising at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, and 648 in a sample of human monocytes is indicative of sepsis. In some embodiments, an increase in expression of one or more target RNAs comprising at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, and 632 in a sample of human monocytes is indicative of sepsis. In some embodiments, an increase in expression of one or more target RNAs comprising at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, and 352 in a sample of human monocytes is indicative of sepsis. In some embodiments, an increase in expression of one or more target RNAs comprising at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 231, 236, 242, 260, 261, 266, and 287 in a sample of human monocytes is indicative of sepsis.
4.1.2. Exemplary Controls
In some embodiments, a normal level (a “control”) for each target RNA can be determined as an average level or range that is characteristic of normal human monocytes or other reference material, against which the level measured in the sample can be compared. The determined average or range of target RNA in normal subjects can be used as a benchmark for detecting above-normal or below-normal levels of target RNA indicative of sepsis. In some embodiments, normal levels of target RNA can be determined using individual or pooled RNA-containing samples from one or more individuals, such as from healthy individuals or from intensive care patients with similar clinical severity of disease (e.g., having matched ICU clinical (APACHE II) scores) to those diagnosed with sepsis syndrome, but without diagnosis of sepsis syndrome.
In some embodiments, determining a normal level of expression of a target RNA comprises detecting a complex comprising a probe hybridized to a nucleic acid selected from a target RNA, a DNA amplicon of the target RNA, and a complement of the target RNA. That is, in some embodiments, a normal level of expression can be determined by detecting a DNA amplicon of the target RNA, or a complement of the target RNA rather than the target RNA itself. In some embodiments, a normal level of such a complex is determined and used as a control. The normal level of the complex, in some embodiments, correlates to the normal level of the target RNA. Thus, when a normal level of a target is discussed herein, that level can, in some embodiments, be determined by detecting such a complex.
In some embodiments, a control comprises RNA from cells of a single individual, e.g., a healthy individual or an intensive care patient with similar clinical severity of disease (e.g., having matched ICU clinical (APACHE II) scores) to a patient being tested for sepsis, but without diagnosis of sepsis syndrome. In some embodiments, a control comprises RNA from a pool of cells from multiple individuals. In some embodiments, a control comprises commercially-available human RNA, such as, for example, total RNA from CD14+ cells. In some embodiments, a normal level or normal range has already been predetermined prior to testing a sample for an elevated level.
In some embodiments, the normal level of target RNA can be determined from one or more continuous cell lines, typically cell lines previously shown to have expression levels of the at least one target RNA that approximate the level of expression in normal human monocytes.
In some embodiments, a method comprises detecting the level of expression of at least one target RNA. In some embodiments, a method further comprises comparing the level of expression of at least one target RNA to a normal level of expression of the at least one target RNA. In some embodiments, a method further comprises comparing the level of expression of at least one target RNA to a control level of expression of the at least one target RNA. A control level of expression of the at least one target RNA is, in some embodiments, the level of expression of the at least one target RNA in a normal cell. In some such embodiments, a control level may be referred to as a normal level. In some embodiments, a greater level of expression of the at least one target RNA relative to the level of expression of the at least one target RNA in a normal cell indicates sepsis. In some embodiments, a reduced level of expression of the at least one target RNA relative to the level of expression of the at least one target RNA in a normal cell indicates sepsis.
In some embodiments, the level of expression of the at least one target RNA is compared to a reference level of expression, e.g., from a patient with a confirmed case of sepsis syndrome. In some such embodiments, a similar level of expression of the at least one target RNA relative to the reference sample indicates sepsis.
In some embodiments, a level of expression of at least one target RNA that is at least about two-fold greater than a normal level of expression of the respective at least one target RNA indicates the presence of sepsis. In some embodiments, a level of expression of at least one target RNA that is at least about two-fold greater than the level of the respective at least one target RNA in a control sample comprised of normal cells indicates the presence of a sepsis. In various embodiments, a level of expression of at least one target RNA that is at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold greater than the level of expression of the respective at least one target RNA in a control sample comprised of normal cells indicates the presence of sepsis. In various embodiments, a level of expression of at least one target RNA that is at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold greater than a normal level of expression of the at least one target RNA indicates the presence of sepsis.
In some embodiments, a level of expression of at least one target RNA that is reduced by at least about two-fold relative to a normal level of expression of the respective at least one target RNA indicates the presence of sepsis. In some embodiments, a level of expression of at least one target RNA that is reduced by at least about two-fold as compared to the level of the respective at least one target RNA in a control sample comprised of normal cells indicates the presence of a sepsis. In various embodiments, a level of expression of at least one target RNA that is reduced by at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold as compared to the level of expression of the respective at least one target RNA in a control sample comprised of normal cells indicates the presence of sepsis. In various embodiments, a level of expression of at least one target RNA that is reduced by at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold as compared to a normal level of expression of the at least one target RNA indicates the presence of sepsis.
In some embodiments, a control level of expression of a target RNA is determined contemporaneously, such as in the same assay or batch of assays, as the level of expression of the target RNA in a sample. In some embodiments, a control level of expression of a target RNA is not determined contemporaneously as the level of expression of the target RNA in a sample. In some such embodiments, the control level of expression has been determined previously.
In some embodiments, the level of expression of a target RNA is not compared to a control level of expression, for example, when it is known that the target RNA is expressed at very low levels, or not at all, in normal cells. In such embodiments, detection of a high level of the target RNA in a sample is indicative of sepsis.
4.1.3. Exemplary Methods of Preparing RNAs
Target RNA can be prepared by any appropriate method. Total RNA can be isolated by any method, including, but not limited to, the protocols set forth in Wilkinson, M. (1988) Nucl. Acids Res. 16(22):10, 933; and Wilkinson, M. (1988) Nucl. Acids Res. 16(22): 10934, or by using commercially-available kits or reagents, such as the TRIzol® reagent (InvitrogenTM), Total RNA Extraction Kit (iNtRON Biotechnology), Total RNA Purification Kit (Norgen Biotek Corp.), RNAqueousTM (Ambion), MagMAXTM (Ambion), RecoverAllTM (Ambion), RNeasy (Qiagen), etc.
In some embodiments, small RNAs are isolated or enriched. In some embodiments “small RNA” refers to RNA molecules smaller than about 200 nucleotides (nt) in length. In some embodiments, “small RNA” refers to RNA molecules smaller than about 100 nt, smaller than about 90 nt, smaller than about 80 nt, smaller than about 70 nt, smaller than about 60 nt, smaller than about 50 nt, or smaller than about 40 nt.
Enrichment of small RNAs can be accomplished by method. Such methods include, but are not limited to, methods involving organic extraction followed by adsorption of nucleic acid molecules on a glass fiber filter using specialized binding and wash solutions, and methods using spin column purification. Enrichment of small RNAs may be accomplished using commercially-available kits, such as mirVanaTM Isolation Kit (Applied Biosystems), mirPremierTM microRNA Isolation Kit (Sigma-Aldrich), PureLinkTM miRNA Isolation Kit (Invitrogen), miRCURYTM RNA isolation kit (Exiqon), microRNA Purification Kit (Norgen Biotek Corp.), miRNeasy kit (Qiagen), etc. In some embodiments, purification can be accomplished by the TRIzol® (Invitrogen) method, which employs a phenol/isothiocyanate solution to which chloroform is added to separate the RNA-containing aqueous phase. Small RNAs are subsequently recovered from the aqueous by precipitation with isopropyl alcohol. In some embodiments, small RNAs can be purified using chromatographic methods, such as gel electrophoresis using the flashPAGETM Fractionator available from Applied Biosystems.
In some embodiments, small RNA is isolated from other RNA molecules to enrich for target RNAs, such that the small RNA fraction (e.g., containing RNA molecules that are 200 nucleotides or less in length, such as less than 100 nucleotides in length, such as less than 50 nucleotides in length, such as from about 10 to about 40 nucleotides in length) is substantially pure, meaning it is at least about 80%, 85%, 90%, 95% pure or more, but less than 100% pure, with respect to larger RNA molecules. Alternatively, enrichment of small RNA can be expressed in terms of fold-enrichment. In some embodiments, small RNA is enriched by about, at least about, or at most about 5×, 10×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 110×, 120×, 130×, 140×, 150×, 160×, 170×, 180×, 190×, 200×, 210×, 220×, 230×, 240×, 250×, 260×, 270×, 280×, 290×, 300×, 310×, 320×, 330×, 340×, 350×, 360×, 370×, 380×, 390×, 400×, 410×, 420×, 430×, 440×, 450×, 460×, 470×, 480×, 490×, 500×, 600×, 700×, 800×, 900×, 1000×, 1100×, 1200×, 1300×, 1400×, 1500×, 1600×, 1700×, 1800×, 1900×, 2000×, 3000×, 4000×, 5000×, 6000×, 7000×, 8000×, 9000×, 10,000× or more, or any range derivable therein, with respect to the concentration of larger RNAs in an RNA isolate or total RNA in a sample.
In yet other embodiments, expression is measured in a sample in which RNA has not first been purified from the cells.
In some embodiments, RNA is modified before target RNAs are detected. In some embodiments, the modified RNA is total RNA. In other embodiments, the modified RNA is small RNA that has been purified from total RNA or from cell lysates, such as RNA less than 200 nucleotides in length, such as less than 100 nucleotides in length, such as less than 50 nucleotides in length, such as from about 10 to about 40 nucleotides in length. RNA modifications that can be utilized in the methods described herein include, but are not limited to, the addition of a poly-dA or a poly-dT tail, which can be accomplished chemically or enzymatically, and/or the addition of a small molecule, such as biotin.
In some embodiments, one or more target RNAs are reverse transcribed. In some embodiments, where present, RNA is modified when it is reverse transcribed, such as when a poly-dA or a poly-dT tail is added to the cDNA during reverse transcription. In other embodiments, RNA is modified before it is reverse transcribed. In some embodiments, total RNA is reverse transcribed. In other embodiments, small RNAs are isolated or enriched before the RNA is reverse transcribed.
When a target RNA is reverse transcribed, a complement of the target RNA is formed. In some embodiments, the complement of the target RNA is detected rather than the target RNA itself (or a DNA copy thereof). Thus, when the methods discussed herein indicate that a target RNA is detected, or the level of a target RNA is determined, such detection or determination may be carried out on a complement of the target RNA instead of or in addition to, the target RNA itself. In some embodiments, when the complement of the target RNA is detected rather than the target RNA, a probe is used that is complementary to the complement of the target RNA. In such embodiments, the probe comprises at least a portion that is identical in sequence to the target RNA, although it may contain thymidine in place of uridine, and/or comprise other modified nucleotides.
In some embodiments, the method of detecting one or more target RNAs comprises amplifying cDNA complementary to said target RNA. Such amplification can be accomplished by any method. Exemplary methods include, but are not limited to, real time PCR, endpoint PCR, and amplification using T7 polymerase from a T7 promoter annealed to a cDNA, such as provided by the SenseAmp PlusTM Kit available at Implen, Germany.
When a target RNA or a cDNA complementary to a target RNA is amplified, in some embodiments, a DNA amplicon of a target RNA is formed. A DNA amplicon may be single stranded or double-stranded. In some embodiments, when a DNA amplicon is single-stranded, the sequence of the DNA amplicon is related to the target RNA in either the sense or antisense orientation. In some embodiments, the DNA amplicon of the target RNA is detected rather than the target RNA itself. Thus, when the methods discussed herein indicate that a target RNA is detected, or the level of a target RNA is determined, such detection or determination may be carried out on a DNA amplicon of the target RNA instead of, or in addition to, the target RNA itself. In some embodiments, when the DNA amplicon of the target RNA is detected rather than the target RNA, a probe is used that is complementary to the complement of the target RNA. In some embodiments, when the DNA amplicon of the target RNA is detected rather than the target RNA, a probe is used that is complementary to the target RNA. Further, I some embodiments, multiple probes may be used, and some probes may be complementary to the target RNA and some probes may be complementary to the complement of the target RNA.
In some embodiments, the method of detecting one or more target RNAs comprises RT-PCR, as described below. In some embodiments, detecting one or more target RNAs comprises real-time monitoring of an RT-PCR reaction, which can be accomplished by any method. Such methods include, but are not limited to, the use of TaqMan®, Molecular beacon, or Scorpion probes (i.e., FRET probes) and the use of intercalating dyes, such as SYBR green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, etc.
4.1.4. Exemplary Analytical Methods
As described above, methods are presented for detecting sepsis in a sample from a patient. In some embodiments, the method comprises detecting a level of expression of at least one target RNA capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1 to 86 set forth in Table 2 that is greater in the sample than a normal level of expression of the at least one target RNA in a control sample, such as a sample from a patient that has not been diagnosed with sepsis syndrome, or a sample of normal human monocytes. In some embodiments, a method comprises detecting a level of one or more target RNAs that comprise a sequence that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86 that is greater in the sample than a normal level of expression of the at least one target RNA in a control sample. In some embodiments, a method comprises detecting a level of one or more target RNAs that comprise at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897 that is greater in the sample than a normal level of expression of the at least one target RNA in a control sample. In some embodiments, a target RNA, in its mature form, comprises fewer than 30 nucleotides. In some embodiments, a target RNA is a microRNA.
In some embodiments, such as those described above, the method further comprises detecting a level of expression of at least one target RNA of the human miRNome that does not specifically hybridize to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1 to 86 and does not comprise at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897, that is greater in the sample than a normal level of expression of the at least one target RNA in a control sample. As used herein, the term “human miRNome” refers to all microRNA genes in a human cell and the mature microRNAs produced therefrom.
Any analytical procedure capable of permitting specific and quantifiable (or semi-quantifiable) detection of the desired at least one target RNA may be used in the methods herein presented. Such analytical procedures include, but are not limited to, the microarray methods set forth in Example 1, the microbead methods set forth in Example 2, and methods known to those skilled in the art.
In some embodiments, detection of a target RNA comprises forming a complex comprising a polynucleotide that is complementary to a target RNA or to a complement thereof, and a nucleic acid selected from the target RNA, a DNA amplicon of the target RNA, and a complement of the target RNA. Thus, in some embodiments, the polynucleotide forms a complex with a target RNA. In some embodiments, the polynucleotide forms a complex with a complement of the target RNA, such as a cDNA that has been reverse transcribed from the target RNA. In some embodiments, the polynucleotide forms a complex with a DNA amplicon of the target RNA. When a double-stranded DNA amplicon is part of a complex, as used herein, the complex may comprise one or both strands of the DNA amplicon. Thus, in some embodiments, a complex comprises only one strand of the DNA amplicon. In some embodiments, a complex is a triplex and comprises the polynucleotide and both strands of the DNA amplicon. In some embodiments, the complex is formed by hybridization between the polynucleotide and the target RNA, complement of the target RNA, or DNA amplicon of the target RNA. The polynucleotide, in some embodiments, is a primer or probe.
In some embodiments, a method comprises detecting the complex. In some embodiments, the complex does not have to be associated at the time of detection. That is, in some embodiments, a complex is formed, the complex is then dissociated or destroyed in some manner, and components from the complex are detected. An example of such a system is a TaqMan® assay. In some embodiments, when the polynucleotide is a primer, detection of the complex may comprise amplification of the target RNA, a complement of the target RNA, or a DNA amplicon of a target RNA.
In some embodiments the analytical method used for detecting at least one target RNA in the methods set forth herein includes real-time quantitative RT-PCR. See Chen, C. et al. (2005) Nucl. Acids Res. 33:e179 and PCT Publication No. WO 2007/117256, which are incorporated herein by reference in its entirety. In some embodiments, the analytical method used for detecting at least one target RNA includes the method described in U.S. Publication No. US2009/0123912 A1, which is incorporated herein by reference in its entirety. In an exemplary method described in that publication, an extension primer comprising a first portion and second portion, wherein the first portion selectively hybridizes to the 3′ end of a particular microRNA and the second portion comprises a sequence for universal primer, is used to reverse transcribe the microRNA to make a cDNA. A reverse primer that selectively hybridizes to the 5′ end of the microRNA and a universal primer are then used to amplify the cDNA in a quantitative PCR reaction.
In some embodiments, the analytical method used for detecting at least one target RNA includes the use of a TaqMan® probe. In some embodiments, the analytical method used for detecting at least one target RNA includes a TaqMan® assay, such as the TaqMan® MicroRNA Assays sold by Applied Biosystems, Inc. In an exemplary TaqMan® assay, total RNA is isolated from the sample. In some embodiments, the assay can be used to analyze about 10 ng of total RNA input sample, such as about 9 ng of input sample, such as about 8 ng of input sample, such as about 7 ng of input sample, such as about 6 ng of input sample, such as about 5 ng of input sample, such as about 4 ng of input sample, such as about 3 ng of input sample, such as about 2 ng of input sample, and even as little as about 1 ng of input sample containing microRNAs.
The TaqMan® assay utilizes a stem-loop primer that is specifically complementary to the 3′-end of a target RNA. In an exemplary TaqMan® assay, hybridizing the stem-loop primer to the target RNA is followed by reverse transcription of the target RNA template, resulting in extension of the 3′ end of the primer. The result of the reverse transcription is a chimeric (DNA) amplicon with the step-loop primer sequence at the 5′ end of the amplicon and the cDNA of the target RNA at the 3′ end. Quantitation of the target RNA is achieved by real time RT-PCR using a universal reverse primer having a sequence that is complementary to a sequence at the 5′ end of all stem-loop target RNA primers, a target RNA-specific forward primer, and a target RNA sequence-specific TaqMan® probe.
The assay uses fluorescence resonance energy transfer (“FRET”) to detect and quantitate the synthesized PCR product. Typically, the TaqMan® probe comprises a fluorescent dye molecule coupled to the 5′-end and a quencher molecule coupled to the 3′-end, such that the dye and the quencher are in close proximity, allowing the quencher to suppress the fluorescence signal of the dye via FRET. When the polymerase replicates the chimeric amplicon template to which the TaqMan® probe is bound, the 5′-nuclease of the polymerase cleaves the probe, decoupling the dye and the quencher so that FRET is abolished and a fluorescence signal is generated. Fluorescence increases with each RT-PCR cycle proportionally to the amount of probe that is cleaved.
Additional exemplary methods for RNA detection and/or quantification are described, e.g., in U.S. Publication No. US 2007/0077570 (Lao et al.), PCT Publication No. WO 2007/025281 (Tan et al.), U.S. Publication No. US2007/0054287 (Bloch), PCT Publication No. WO2006/0130761 (Bloch), and PCT Publication No. WO 2007/011903 (Lao et al.), which are incorporated by reference herein in their entireties for any purpose.
In some embodiments, quantitation of the results of real-time RT-PCR assays is done by constructing a standard curve from a nucleic acid of known concentration and then extrapolating quantitative information for target RNAs of unknown concentration. In some embodiments, the nucleic acid used for generating a standard curve is an RNA (e.g., microRNA) of known concentration. In some embodiments, the nucleic acid used for generating a standard curve is a purified double-stranded plasmid DNA or a single-stranded DNA generated in vitro.
In some embodiments, where the amplification efficiencies of the target nucleic acids and the endogenous reference are approximately equal, quantitation is accomplished by the comparative Ct (cycle threshold, e.g., the number of PCR cycles required for the fluorescence signal to rise above background) method. Ct values are inversely proportional to the amount of nucleic acid target in a sample. In some embodiments, Ct values of the target RNA of interest can be compared with a control or calibrator, such as RNA (e.g., microRNA) from normal tissue. In some embodiments, the Ct values of the calibrator and the target RNA samples of interest are normalized to an appropriate endogenous housekeeping gene.
In addition to the TaqMan® assays, other real-time RT-PCR chemistries useful for detecting and quantitating PCR products in the methods presented herein include, but are not limited to, Molecular Beacons, Scorpion probes and intercalating dyes, such as SYBR Green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, etc., which are discussed below.
In some embodiments, real-time RT-PCR detection is performed specifically to detect and quantify the expression of a single target RNA. The target RNA, in some embodiments, is selected from a target RNA capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, the target RNA specifically hybridizes to a nucleic acid comprising a sequence selected from SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 and 86. In some embodiments, the target RNA specifically hybridizes to a nucleic acid comprising a sequence selected from SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 and 79. In some embodiments, the target RNA specifically hybridizes to a nucleic acid comprising a sequence selected from SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 and 84. In some embodiments, the target RNA specifically hybridizes to a nucleic acid comprising a sequence selected from SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, and 78. In some embodiments, the target RNA specifically hybridizes to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 and 85. In some embodiments, the target RNA specifically hybridizes to a nucleic acid comprising a sequence selected from SEQ ID NOs: 9, 50, 51, 70, 72 and 75. In some embodiments, the target RNA comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, the target RNA comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, and 648. In some embodiments, the target RNA comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868. In some embodiments, the target RNA comprises a sequence that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, a target RNA, in its mature form, comprises fewer than 30 nucleotides. In some embodiments, a target RNA is a microRNA.
In various embodiments, real-time RT-PCR detection is utilized to detect, in a single multiplex reaction, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 target RNAs. At least one target RNA, in some embodiments, is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, at least one target RNA comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, at least one target RNA comprises a sequence that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, a target RNA, in its mature form, comprises fewer than 30 nucleotides. In some embodiments, a target RNA is a microRNA.
In some embodiments, the method comprises detecting expression in a multiplex RT-PCR reaction of at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 target RNAs, wherein each target RNA is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 and 86. In some embodiments, the method comprises detecting greater than normal expression, using a single multiplex RT-PCR reaction, of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, or at least 15 target RNAs, wherein each target RNA is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 and 79. In some embodiments, the method comprises detecting greater than normal expression, using a single multiplex RT-PCR reaction, of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, or at least 15 target RNAs, wherein each target RNA is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 and 84. In some embodiments, the method comprises detecting expression in a multiplex RT-PCR reaction of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 target RNAs, wherein each target RNA is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, and 78. In some embodiments, the method comprises detecting expression in a multiplex RT-PCR reaction of at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, or at least 30 target RNAs, wherein each target RNA is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 and 85. In some embodiments, the method comprises detecting expression in a multiplex RT-PCR reaction of at least 2, at least 3, at least 4, at least 5, or at least 6 target RNAs, wherein each target RNA is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 9, 50, 51, 70, 72 and 75. In some embodiments, the method comprises detecting expression in a multiplex RT-PCR reaction of at least two, at least five, at least 10, at least 15, at least 20, at least 25, or at least 30 target RNAs, wherein each target RNA is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, and 648. In some embodiments, the method comprises detecting expression in a multiplex RT-PCR reaction of at least two, at least five, at least 10, at least 15, or at least 20 target RNAs, wherein each target RNA is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, and 632. In some embodiments, the method comprises detecting expression in a multiplex RT-PCR reaction of at least two, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, or at least 70 target RNAs, wherein each target RNA is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 226 to 289, 565 to 604, and 863 to 868.
In some multiplex embodiments, a plurality of probes, such as TaqMan®, probes, each specific for a different RNA target, is used. In some embodiments, each target RNA-specific probe is spectrally distinguishable from the other probes used in the same multiplex reaction.
In some embodiments, quantitation of real-time RT PCR products is accomplished using a dye that binds to double-stranded DNA products, such as SYBR Green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, etc. In some embodiments, the assay is the QuantiTect SYBR Green PCR assay from Qiagen. In this assay, total RNA is first isolated from a sample. Total RNA is subsequently poly-adenylated at the 3′-end and reverse transcribed using a universal primer with poly-dT at the 5′-end. In some embodiments, a single reverse transcription reaction is sufficient to assay multiple target RNAs. Real-time RT-PCR is then accomplished using target RNA-specific primers and an miScript Universal Primer, which comprises a poly-dT sequence at the 5′-end. SYBR Green dye binds non-specifically to double-stranded DNA and upon excitation, emits light. In some embodiments, buffer conditions that promote highly-specific annealing of primers to the PCR template (e.g., available in the QuantiTect SYBR Green PCR Kit from Qiagen) can be used to avoid the formation of non-specific DNA duplexes and primer dimers that will bind SYBR Green and negatively affect quantitation. Thus, as PCR product accumulates, the signal from SYBR Green increases, allowing quantitation of specific products.
Real-time RT-PCR is performed using any RT-PCR instrumentation available in the art. Typically, instrumentation used in real-time RT-PCR data collection and analysis comprises a thermal cycler, optics for fluorescence excitation and emission collection, and optionally a computer and data acquisition and analysis software.
In some embodiments, the analytical method used in the methods described herein is a DASL® (cDNA-mediated Annealing, Selection, Extension, and Ligation) Assay, such as the MicroRNA Expression Profiling Assay available from Illumina, Inc. (See www.illumina.com/downloads/MicroRNAAssayWorkflow.pdf). In some embodiments, total RNA is isolated from a sample to be analyzed by any method. Additionally, in some embodiments, small RNAs are isolated from a sample to be analyzed by any method. Total RNA or isolated small RNAs may then be polyadenylated (>18 A residues are added to the 3′-ends of the RNAs in the reaction mixture). The RNA is reverse transcribed using a biotin-labeled DNA primer that comprises from the 5′ to the 3′ end, a sequence that includes a PCR primer site and a poly-dT region that binds to the poly-dA tail of the sample RNA. The resulting biotinylated cDNA transcripts are then hybridized to a solid support via a biotin-streptavidin interaction and contacted with one or more target RNA-specific polynucleotides. The target RNA-specific polynucleotides comprise, from the 5′-end to the 3′-end, a region comprising a PCR primer site, region comprising an address sequence, and a target RNA-specific sequence.
In some DASL® embodiments, the target RNA-specific sequence comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides having a sequence identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, the target RNA-specific sequence comprises a probe sequence that is complementary to at least a portion of a microRNA of the human miRNome.
After hybridization, the target RNA-specific polynucleotide is extended, and the extended products are then eluted from the immobilized cDNA array. A second PCR reaction using a fluorescently-labeled universal primer generates a fluorescently-labeled DNA comprising the target RNA-specific sequence. The labeled PCR products are then hybridized to a microbead array for detection and quantitation.
In some embodiments, the analytical method used for detecting and quantifying the expression of the at least one target RNA in the methods described herein is a bead-based flow cytometric assay. See Lu J. et al. (2005) Nature 435:834-838, which is incorporated herein by reference in its entirety. An example of a bead-based flow cytometric assay is the xMAP® technology of Luminex, Inc. (See www.luminexcorp.com/technology/index.html). In some embodiments, total RNA is isolated from a sample and is then labeled with biotin. The labeled RNA is then hybridized to target RNA-specific capture probes (e.g., FlexmiRTM products sold by Luminex, Inc. at www.luminexcorp.com/products/assays/index.html) that are covalently bound to microbeads, each of which is labeled with 2 dyes having different fluorescence intensities. A streptavidin-bound reporter molecule (e.g., streptavidin-phycoerythrin, also known as “SAPE”) is attached to the captured target RNA and the unique signal of each bead is read using flow cytometry. In some embodiments, the RNA sample (total RNA or enriched small RNAs) is first polyadenylated, and is subsequently labeled with a biotinylated 3DNATM dendrimer (i.e., a multiple-arm DNA with numerous biotin molecules bound thereto), such as those sold by Marligen Biosciences as the VantageTM microRNA Labeling Kit, using a bridging polynucleotide that is complementary to the 3′-end of the poly-dA tail of the sample RNA and to the 5′-end of the polynucleotide attached to the biotinylated dendrimer. The streptavidin-bound reporter molecule is then attached to the biotinylated dendrimer before analysis by flow cytometry. See www.marligen.com/vantage-microrna-labeling-kit.html. In some embodiments, biotin-labeled RNA is first exposed to SAPE, and the RNA/SAPE complex is subsequently exposed to an anti-phycoerythrin antibody attached to a DNA dendrimer, which can be bound to as many as 900 biotin molecules. This allows multiple SAPE molecules to bind to the biotinylated dendrimer through the biotin-streptavidin interaction, thus increasing the signal from the assay.
In some embodiments, the analytical method used for detecting and quantifying the expression of the at least one target RNA in the methods described herein is by gel electrophoresis and detection with labeled probes (e.g., probes labeled with a radioactive or chemiluminescent label), such as by Northern blotting. In some embodiments, total RNA is isolated from the sample, and then is size-separated by SDS polyacrylamide gel electrophoresis. The separated RNA is then blotted onto a membrane and hybridized to radiolabeled complementary probes. In some embodiments, exemplary probes contain one or more affinity-enhancing nucleotide analogs as discussed below, such as locked nucleic acid (“LNA”) analogs, which contain a bicyclic sugar moiety instead of deoxyribose or ribose sugars. See, e.g., Várallyay, E. et al. (2008) Nature Protocols 3(2):190-196, which is incorporated herein by reference in its entirety. In some embodiments, the total RNA sample can be further purified to enrich for small RNAs. In some embodiments, target RNAs can be amplified by, e.g., rolling circle amplification using a long probe that is complementary to both ends of a target RNA (“padlocked probes”), ligation to circularize the probe followed by rolling circle replication using the target RNA hybridized to the circularized probe as a primer. See, e.g., Jonstrup, S. P. et al. (2006) RNA 12:1-6, which is incorporated herein by reference in its entirety. The amplified product can then be detected and quantified using, e.g., gel electrophoresis and Northern blotting.
In alternative embodiments, labeled probes are hybridized to isolated total RNA in solution, after which the RNA is subjected to rapid ribonuclease digestion of single-stranded RNA, e g, unhybridized portions of the probes or unhybridized target RNAs. In these embodiments, the ribonuclease treated sample is then analyzed by SDS-PAGE and detection of the radiolabeled probes by, e.g., Northern blotting. See mirVanaTM miRNA Detection Kit sold by Applied Biosystems, Inc. product literature at www.ambion.com/catalog/CatNum.php?1552.
In some embodiments, the analytical method used for detecting and quantifying the at least one target RNA in the methods described herein is by hybridization to a microarray. See, e.g., Liu, C. G. et al. (2004) Proc. Nat'l Acad. Sci. USA 101:9740-9744; Lim, L. P. et al. (2005) Nature 433:769-773, each of which is incorporated herein by reference in its entirety, and Example 1.
In some embodiments, detection and quantification of a target RNA using a microarray is accomplished by surface plasmon resonance. See, e.g., Nanotech News (2006), available nano.cancer.gov/news_center/nanotech_news_2006-10-30b.asp. In these embodiments, total RNA is isolated from a sample being tested. Optionally, the RNA sample is further purified to enrich the population of small RNAs. After purification, the RNA sample is bound to an addressable microarray containing probes at defined locations on the microarray. Nonlimiting exemplary probes include probes comprising sequences set forth in SEQ ID NOs: 1 to 86. Exemplary probes also include, but are not limited to, probes comprising a region that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 868 to 897. Exemplary probes also include, but are not limited to, probes comprising at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, the probes contain one or more affinity-enhancing nucleotide analogs as discussed below, such as locked nucleic acid (“LNA”) nucleotide analogs. After hybridization to the microarray, the RNA that is hybridized to the array is first polyadenylated, and the array is then exposed to gold particles having poly-dT bound to them. The amount of bound target RNA is quantitated using surface plasmon resonance.
In some embodiments, microarrays are utilized in a RNA-primed, Array-based Klenow Enzyme (“RAKE”) assay. See Nelson, P. T. et al. (2004) Nature Methods 1(2):1-7; Nelson, P. T. et al. (2006) RNA 12(2):1-5, each of which is incorporated herein by reference in its entirety. In some embodiments, total RNA is isolated from a sample. In some embodiments, small RNAs are isolated from a sample. The RNA sample is then hybridized to DNA probes immobilized at the 5′-end on an addressable array. The DNA probes comprise, in some embodiments, from the 5′-end to the 3′-end, a first region comprising a “spacer” sequence which is the same for all probes, a second region comprising three thymidine-containing nucleosides, and a third region comprising a sequence that is complementary to a target RNA of interest.
Exemplary target RNAs of interest include, but are not limited to, target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1 to 86, target RNAs comprising a region that is identical to at least 15 contiguous nucleotides of a sequence selected from 196 to 399, 950, 565 to 707, and 863 to 897, and target RNAs comprising a region that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86. Target RNAs also include target RNAs in the miRNome that do not specifically hybridize to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, a target RNA, in its mature form, comprises fewer than 30 nucleotides. In some embodiments, a target RNA is a microRNA.
After the sample is hybridized to the array, it is exposed to exonuclease I to digest any unhybridized probes. The Klenow fragment of DNA polymerase I is then applied along with biotinylated dATP, allowing the hybridized target RNAs to act as primers for the enzyme with the DNA probe as template. The slide is then washed and a streptavidin-conjugated fluorophore is applied to detect and quantitate the spots on the array containing hybridized and Klenow-extended target RNAs from the sample.
In some embodiments, the RNA sample is reverse transcribed. In some embodiments, the RNA sample is reverse transcribed using a biotin/poly-dA random octamer primer. When than primer is used, the RNA template is digested and the biotin-containing cDNA is hybridized to an addressable microarray with bound probes that permit specific detection of target RNAs. In some embodiments, the microarray includes at least one probe comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides identically present in, or complementary to a region of, a sequence selected from SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897. After hybridization of the cDNA to the microarray, the microarray is exposed to a streptavidin-bound detectable marker, such as a fluorescent dye, and the bound cDNA is detected. See Liu C. G. et al. (2008) Methods 44:22-30, which is incorporated herein by reference in its entirety.
In some embodiments, target RNAs are detected and quantified in an ELISA-like assay using probes bound in the wells of microtiter plates. See Mora J. R. and Getts R. C. (2006) BioTechniques 41:420-424 and supplementary material in BioTechniques 41(4):1-5; U.S. Patent Publication No. 2006/0094025 to Getts et al., each of which is incorporated by reference herein in its entirety. In these embodiments, a sample of RNA that is enriched in small RNAs is either polyadenylated, or is reverse transcribed and the cDNA is polyadenylated. The RNA or cDNA is hybridized to probes immobilized in the wells of a microtiter plates, wherein each of the probes comprises a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897, or a sequence such as one or more sequences of target RNAs (or the reverse complement thereof) of the human miRNome, depending on whether RNA or cDNA is hybridized to the array. In some embodiments, the hybridized RNAs are labeled using a capture sequence, such as a DNA dendrimer (such as those available from Genisphere, Inc., www.genisphere.com/about_3dna.html) that is labeled with a plurality of biotin molecules or with a plurality of horseradish peroxidase molecules, and a bridging polynucleotide that contains a poly-dT sequence at the 5′-end that binds to the poly-dA tail of the captured nucleic acid, and a sequence at the 3′-end that is complementary to a region of the capture sequence. If the capture sequence is biotinylated, the microarray is then exposed to streptavidin-bound horseradish peroxidase. Hybridization of target RNAs is detected by the addition of a horseradish peroxidase substrate such as tetramethylbenzidine (TMB) and measurement of the absorbance of the solution at 450 nM.
In still other embodiments, an addressable microarray is used to detect a target RNA using quantum dots. See Liang, R. Q. et al. (2005) Nucl. Acids Res. 33(2):e17, available at www.pubmedcentral.nih.gov/articlerender.fcgi?artid=548377, which is incorporated herein by reference in its entirety. In some embodiments, total RNA is isolated from a sample. In some embodiments, small RNAs are isolated from the sample. The 3′-ends of the target RNAs are biotinylated using biotin-X-hydrazide. The biotinylated target RNAs are captured on a microarray comprising immobilized probes comprising sequences that are identically present in, or complementary to a region of, one or more of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897 and/or probes comprising sequences other than those that are complementary to one or more microRNAs of the human miRNome. The hybridized target RNAs are then labeled with quantum dots via a biotin-streptavidin binding. A confocal laser causes the quantum dots to fluoresce and the signal can be quantified. In alternative embodiments, small RNAs can be detected using a colorimetric assay. In these embodiments, small RNAs are labeled with streptavidin-conjugated gold followed by silver enhancement. The gold nanoparticles bound to the hybridized target RNAs catalyze the reduction of silver ions to metallic silver, which can then be detected colorimetrically with a CCD camera.
In some embodiments, detection and quantification of one or more target RNAs is accomplished using microfluidic devices and single-molecule detection. In some embodiments, target RNAs in a sample of isolated total RNA are hybridized to two probes, one which is complementary to nucleic acids at the 5′-end of the target RNA and the second which is complementary to the 3′-end of the target RNA. Each probe comprises, in some embodiments, one or more affinity-enhancing nucleotide analogs, such as LNA nucleotide analogs and each is labeled with a different fluorescent dye having different fluorescence emission spectra. The sample is then flowed through a microfluidic capillary in which multiple lasers excite the fluorescent probes, such that a unique coincident burst of photons identifies a particular target RNA, and the number of particular unique coincident bursts of photons can be counted to quantify the amount of the target RNA in the sample. See U.S. Patent Publication No. 2006/0292616 to Neely et al., which is hereby incorporated by reference in its entirety. In some alternative embodiments, a target RNA-specific probe can be labeled with 3 or more distinct labels selected from, e.g., fluorophores, electron spin labels, etc., and then hybridized to an RNA sample, such as total RNA, or a sample that is enriched in small RNAs. Nonlimiting exemplary target RNA-specific probes include probes comprising sequences selected from of SEQ ID NOs: 1 to 86. Nonlimiting exemplary target RNA-specific probes include probes comprising sequences that are complementary to sequences selected from of SEQ ID NOs: 1 to 86. Nonlimiting exemplary target RNA-specific probes also include probes comprising at least 15 contiguous nucleotides of, or the complement of at least 15 contiguous nucleotides of, a sequence selected from SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897.
Optionally, the sample RNA is modified before hybridization. The target RNA/probe duplex is then passed through channels in a microfluidic device and that comprise detectors that record the unique signal of the 3 labels. In this way, individual molecules are detected by their unique signal and counted. See U.S. Pat. Nos. 7,402,422 and 7,351,538 to Fuchs et al., U.S. Genomics, Inc., each of which is incorporated herein by reference in its entirety.
In some embodiments, the detection and quantification of one or more target RNAs is accomplished by a solution-based assay, such as a modified Invader assay. See Allawi H. T. et al. (2004) RNA 10:1153-1161, which is incorporated herein by reference in its entirety. In some embodiments, the modified invader assay can be performed on unfractionated detergent lysates of cells. In other embodiments, the modified invader assay can be performed on total RNA isolated from cells or on a sample enriched in small RNAs. The target RNAs in a sample are annealed to two probes which form hairpin structures. A first probe has a hairpin structure at the 5′ end and a region at the 3′-end that has a sequence that is complementary to the sequence of a region at the 5′-end of a target RNA. The 3′-end of the first probe is the “invasive polynucleotide”. A second probe has, from the 5′ end to the 3′-end a first “flap” region that is not complementary to the target RNA, a second region that has a sequence that is complementary to the 3′-end of the target RNA, and a third region that forms a hairpin structure. When the two probes are bound to a target RNA target, they create an overlapping configuration of the probes on the target RNA template, which is recognized by the Cleavase enzyme, which releases the flap of the second probe into solution. The flap region then binds to a complementary region at the 3′-end of a secondary reaction template (“SRT”). A FRET polynucleotide (having a fluorescent dye bound to the 5′-end and a quencher that quenches the dye bound closer to the 3′ end) binds to a complementary region at the 5′-end of the SRT, with the result that an overlapping configuration of the 3′-end of the flap and the 5′-end of the FRET polynucleotide is created. Cleavase recognizes the overlapping configuration and cleaves the 5′-end of the FRET polynucleotide, generates a fluorescent signal when the dye is released into solution.
4.1.5. Exemplary Polynucleotides
In some embodiments, polynucleotides are provided. In some embodiments, synthetic polynucleotides are provided. Synthetic polynucleotides, as used herein, refer to polynucleotides that have been synthesized in vitro either chemically or enzymatically. Chemical synthesis of polynucleotides includes, but is not limited to, synthesis using polynucleotide synthesizers, such as OligoPilot (GE Healthcare), ABI 3900 DNA Synthesizer (Applied Biosystems), and the like. Enzymatic synthesis includes, but is not limited, to producing polynucleotides by enzymatic amplification, e.g., PCR.
In some embodiments, a polynucleotide is provided that comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897 and sequences complementary to SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, the polynucleotide further comprises a region having a sequence that is not found in, or complementary to, any of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, a polynucleotide is provided that comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 67, 215 to 399, 950, and 863 to 897, and sequences complementary to SEQ ID NOs: 1 to 67, 215 to 399, 950, and 863 to 897. In some embodiments, the polynucleotide further comprises a region having a sequence that is not found in, or complementary to, any of SEQ ID NOs: 1 to 67, 215 to 399, 950, or 863 to 897.
A “region” can comprise the full-length sequence, or the complement of the full-length sequence, of a particular sequence, such as any of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897, or it can comprise a subsequence, or the complement of a subsequence, of a particular sequence, such as any of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897. Such subsequences may comprise, in some embodiments, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more contiguous nucleotides from a particular SEQ ID NO or its complement.
In various embodiments, a polynucleotide comprises fewer than 500, fewer than 300, fewer than 200, fewer than 150, fewer than 100, fewer than 75, fewer than 50, fewer than 40, or fewer than 30 nucleotides. In various embodiments, a polynucleotide is between 8 and 200, between 8 and 150, between 8 and 100, between 8 and 75, between 8 and 50, between 8 and 40, or between 8 and 30 nucleotides long.
In some embodiments, the polynucleotide is a primer. In some embodiments, the primer is labeled with a detectable moiety. In some embodiments, a primer is not labeled. A primer, as used herein, is a polynucleotide that is capable of specifically hybridizing to a target RNA or to a cDNA reverse transcribed from the target RNA or to an amplicon that has been amplified from a target RNA or a cDNA (collectively referred to as “template”), and, in the presence of the template, a polymerase and suitable buffers and reagents, can be extended to form a primer extension product.
In some embodiments, the polynucleotide is a probe. In some embodiments, the probe is labeled with a detectable moiety. A detectable moiety, as used herein, includes both directly detectable moieties, such as fluorescent dyes, and indirectly detectable moieties, such as members of binding pairs. When the detectable moiety is a member of a binding pair, in some embodiments, the probe can be detectable by incubating the probe with a detectable label bound to the second member of the binding pair. In some embodiments, a probe is not labeled, such as when a probe is a capture probe, e.g., on a microarray or bead. In some embodiments, a probe is not extendable, e.g., by a polymerase. In other embodiments, a probe is extendable.
In some embodiments, the polynucleotide is a FRET probe that in some embodiments is labeled at the 5′-end with a fluorescent dye (donor) and at the 3′-end with a quencher (acceptor), a chemical group that absorbs (i.e., suppresses) fluorescence emission from the dye when the groups are in close proximity (i.e., attached to the same probe). In other embodiments, the donor and acceptor are not at the ends of the FRET probe. Thus, in some embodiments, the emission spectrum of the donor moiety should overlap considerably with the absorption spectrum of the acceptor moiety.
4.1.5.1. Exemplary Polynucleotide Modifications
In some embodiments, the methods of detecting at least one target RNA described herein employ one or more polynucleotides that have been modified, such as polynucleotides comprising one or more affinity-enhancing nucleotide analogs. Modified polynucleotides useful in the methods described herein include primers for reverse transcription, PCR amplification primers, and probes. In some embodiments, the incorporation of affinity-enhancing nucleotides increases the binding affinity and specificity of a polynucleotide for its target nucleic acid as compared to polynucleotides that contain only deoxyribonucleotides, and allows for the use of shorter polynucleotides or for shorter regions of complementarity between the polynucleotide and the target nucleic acid.
In some embodiments, affinity-enhancing nucleotide analogs include nucleotides comprising one or more base modifications, sugar modifications and/or backbone modifications.
In some embodiments, modified bases for use in affinity-enhancing nucleotide analogs include 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine, xanthine and hypoxanthine.
In some embodiments, affinity-enhancing nucleotide analogs include nucleotides having modified sugars such as 2′-substituted sugars, such as 2′-O-alkyl-ribose sugars, 2′-amino-deoxyribose sugars, 2′-fluoro-deoxyribose sugars, 2′-fluoro-arabinose sugars, and 2′-O-methoxyethyl-ribose (2′MOE) sugars. In some embodiments, modified sugars are arabinose sugars, or d-arabino-hexitol sugars.
In some embodiments, affinity-enhancing nucleotide analogs include backbone modifications such as the use of peptide nucleic acids (PNA; e.g., an oligomer including nucleobases linked together by an amino acid backbone). Other backbone modifications include phosphorothioate linkages, phosphodiester modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acid, methylphosphonate, alkylphosphonates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof.
In some embodiments, a polynucleotide includes at least one affinity-enhancing nucleotide analog that has a modified base, at least nucleotide (which may be the same nucleotide) that has a modified sugar, and/or at least one internucleotide linkage that is non-naturally occurring.
In some embodiments, an affinity-enhancing nucleotide analog contains a locked nucleic acid (“LNA”) sugar, which is a bicyclic sugar. In some embodiments, a polynucleotide for use in the methods described herein comprises one or more nucleotides having an LNA sugar. In some embodiments, a polynucleotide contains one or more regions consisting of nucleotides with LNA sugars. In other embodiments, a polynucleotide contains nucleotides with LNA sugars interspersed with deoxyribonucleotides. See, e.g., Frieden, M. et al. (2008) Curr. Pham. Des. 14(11):1138-1142.
4.1.5.2. Exemplary Primers
In some embodiments, a primer is provided. In some embodiments, a primer is identical or complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of a target RNA. In some embodiments, a primer may also comprise portions or regions that are not identical or complementary to the target RNA. In some embodiments, a region of a primer that is identical or complementary to a target RNA is contiguous, such that any region of a primer that is not identical or complementary to the target RNA does not disrupt the identical or complementary region.
In some embodiments, a primer comprises a portion that is identically present in a target RNA. In some such embodiments, a primer that comprises a region that is identically present in the target RNA is capable of selectively hybridizing to a cDNA that has been reverse transcribed from the RNA, or to an amplicon that has been produced by amplification of the target RNA or cDNA. In some embodiments, the primer is complementary to a sufficient portion of the cDNA or amplicon such that it selectively hybridizes to the cDNA or amplicon under the conditions of the particular assay being used.
As used herein, “selectively hybridize” means that a polynucleotide, such as a primer or probe, will hybridize to a particular nucleic acid in a sample with at least 5-fold greater affinity than it will hybridize to another nucleic acid present in the same sample that has a different nucleotide sequence in the hybridizing region. Exemplary hybridization conditions are discussed in Example 1. In some embodiments, a polynucleotide will hybridize to a particular nucleic acid in a sample with at least 10-fold greater affinity than it will hybridize to another nucleic acid present in the same sample that has a different nucleotide sequence in the hybridizing region.
Nonlimiting exemplary primers include primers comprising sequences that are identically present in, or complementary to a region of sequences selected from SEQ ID NOs: 1 to 86. Exemplary primers also include, but are not limited to, primers comprising regions that are identical or complementary to at least 15 contiguous nucleotides of sequences selected from SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897.
In some embodiments, a primer is used to reverse transcribe a target RNA, for example, as discussed herein. In some embodiments, a primer is used to amplify a target RNA or a cDNA reverse transcribed therefrom. Such amplification, in some embodiments, is quantitative PCR, for example, as discussed herein. In some embodiments, a primer comprises a detectable moiety.
4.1.5.3. Exemplary Probes
In various embodiments, methods of detecting the presence of a sepsis comprise hybridizing nucleic acids of a human sample with a probe. In some embodiments, the probe comprises a portion that is complementary to a target RNA. In some embodiments, the probe comprises a portion that is identically present in the target RNA. In some such embodiments, a probe that is complementary to a target RNA is complementary to a sufficient portion of the target RNA such that it selectively hybridizes to the target RNA under the conditions of the particular assay being used. In some embodiments, a probe that is complementary to a target RNA is complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of the target RNA. In some embodiments, a probe that is complementary to a target RNA comprises a region that is complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of the target RNA. That is, a probe that is complementary to a target RNA may also comprise portions or regions that are not complementary to the target RNA. In some embodiments, a region of a probe that is complementary to a target RNA is contiguous, such that any region of a probe that is not complementary to the target RNA does not disrupt the complementary region.
In some embodiments, the probe comprises a portion that is identically present in the target RNA. In some such embodiments, a probe that comprises a region that is identically present in the target RNA is capable of selectively hybridizing to a cDNA that has been reverse transcribed from the RNA, or to an amplicon that has been produced by amplification of the target RNA or cDNA. In some embodiments, the probe is complementary to a sufficient portion of the cDNA or amplicon such that it selectively hybridizes to the cDNA or amplicon under the conditions of the particular assay being used. In some embodiments, a probe that is complementary to a cDNA or amplicon is complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of the cDNA or amplicon. In some embodiments, a probe that is complementary to a target RNA comprises a region that is complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of the cDNA or amplicon. That is, a probe that is complementary to a cDNA or amplicon may also comprise portions or regions that are not complementary to the cDNA or amplicon. In some embodiments, a region of a probe that is complementary to a cDNA or amplicon is contiguous, such that any region of a probe that is not complementary to the cDNA or amplicon does not disrupt the complementary region.
Nonlimiting exemplary probes include probes comprising sequences set forth in SEQ ID NOs: 1 to 86. Nonlimiting exemplary probes include probes comprising sequences that are identically present in, or complementary to a region of, sequences selected from SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897. Exemplary probes also include, but are not limited to, probes comprising regions that are identical or complementary to at least 15 contiguous nucleotides of sequences selected from SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897.
In some embodiments, the method of detectably quantifying one or more target RNAs comprises: (a) isolating total RNA; (b) reverse transcribing a target RNA to produce a cDNA that is complementary to the target RNA; (c) amplifying the cDNA from (b); and (d) detecting the amount of a target RNA using real time RT-PCR and a detection probe.
As described above, in some embodiments, the real time RT-PCR detection is performed using a FRET probe, which includes, but is not limited to, a TaqMan® probe, a Molecular beacon probe and a Scorpion probe. In some embodiments, the real time RT-PCR detection and quantification is performed with a TaqMan® probe, i.e., a linear probe that typically has a fluorescent dye covalently bound at one end of the DNA and a quencher molecule covalently bound at the other end of the DNA. The FRET probe comprises a sequence that is complementary to a region of the cDNA such that, when the FRET probe is hybridized to the cDNA, the dye fluorescence is quenched, and when the probe is digested during amplification of the cDNA, the dye is released from the probe and produces a fluorescence signal. In such embodiments, the amount of target RNA in the sample is proportional to the amount of fluorescence measured during cDNA amplification.
The TaqMan® probe typically comprises a region of contiguous nucleotides having a sequence that is complementary to a region of a target RNA or its complementary cDNA that is reverse transcribed from the target RNA template (i.e., the sequence of the probe region is complementary to or identically present in the target RNA to be detected) such that the probe is specifically hybridizable to the resulting PCR amplicon. In some embodiments, the probe comprises a region of at least 6 contiguous nucleotides having a sequence that is fully complementary to or identically present in a region of a cDNA that has been reverse transcribed from a target RNA template, such as comprising a region of at least 8 contiguous nucleotides, at least 10 contiguous nucleotides, at least 12 contiguous nucleotides, at least 14 contiguous nucleotides, or at least 16 contiguous nucleotides having a sequence that is complementary to or identically present in a region of a cDNA reverse transcribed from a target RNA to be detected.
In some embodiments, the region of the cDNA that has a sequence that is complementary to the TaqMan® probe sequence is at or near the center of the cDNA molecule. In some embodiments, there are independently at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides of the cDNA at the 5′-end and at the 3′-end of the region of complementarity.
In some embodiments, Molecular Beacons can be used to detect and quantitate PCR products. Like TaqMan® probes, Molecular Beacons use FRET to detect and quantitate a PCR product via a probe having a fluorescent dye and a quencher attached at the ends of the probe. Unlike TaqMan® probes, Molecular Beacons remain intact during the PCR cycles. Molecular Beacon probes form a stem-loop structure when free in solution, thereby allowing the dye and quencher to be in close enough proximity to cause fluorescence quenching. When the Molecular Beacon hybridizes to a target, the stem-loop structure is abolished so that the dye and the quencher become separated in space and the dye fluoresces. Molecular Beacons are available, e.g., from Gene LinkTM (see www.genelink.com/newsite/products/mbintro.asp).
In some embodiments, Scorpion probes can be used as both sequence-specific primers and for PCR product detection and quantitation. Like Molecular Beacons, Scorpion probes form a stem-loop structure when not hybridized to a target nucleic acid. However, unlike Molecular Beacons, a Scorpion probe achieves both sequence-specific priming and PCR product detection. A fluorescent dye molecule is attached to the 5′-end of the Scorpion probe, and a quencher is attached to the 3′-end. The 3′ portion of the probe is complementary to the extension product of the PCR primer, and this complementary portion is linked to the 5′-end of the probe by a non-amplifiable moiety. After the Scorpion primer is extended, the target-specific sequence of the probe binds to its complement within the extended amplicon, thus opening up the stem-loop structure and allowing the dye on the 5′-end to fluoresce and generate a signal. Scorpion probes are available from, e.g, Premier Biosoft International (see www.premierbiosoft.com/tech_notes/Scorpion.html).
In some embodiments, labels that can be used on the FRET probes include colorimetric and fluorescent labels such as Alexa Fluor dyes, BODIPY dyes, such as BODIPY FL; Cascade Blue; Cascade Yellow; coumarin and its derivatives, such as 7-amino-4-methylcoumarin, aminocoumarin and hydroxycoumarin; cyanine dyes, such as Cy3 and Cy5; eosins and erythrosins; fluorescein and its derivatives, such as fluorescein isothiocyanate; macrocyclic chelates of lanthanide ions, such as Quantum DyeTM; Marina Blue; Oregon Green; rhodamine dyes, such as rhodamine red, tetramethylrhodamine and rhodamine 6G; Texas Red; fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer; and, TOTAB.
Specific examples of dyes include, but are not limited to, those identified above and the following: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750; amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR, and, BODIPY-TR; Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, 2′, 4′,5′,7′-Tetrabromosulfonefluorescein, and TET.
Specific examples of fluorescently labeled ribonucleotides useful in the preparation of RT-PCR probes for use in some embodiments of the methods described herein are available from Molecular Probes (Invitrogen), and these include, Alexa Fluor 488-5-UTP, Fluorescein-12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP, Tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, and BODIPY TR-14-UTP. Other fluorescent ribonucleotides are available from Amersham Biosciences (GE Healthcare), such as Cy3-UTP and Cy5-UTP.
Examples of fluorescently labeled deoxyribonucleotides useful in the preparation of RT-PCR probes for use in the methods described herein include Dinitrophenyl (DNP)-1′-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPY TMR-14-dUTP, Tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630/650-14-dUTP, BODIPY 650/665-14-dUTP; Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor 546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA-dCTP, Alexa Fluor 647-12-OBEA-dCTP. Fluorescently labeled nucleotides are commercially available and can be purchased from, e.g., Invitrogen.
In some embodiments, dyes and other moieties, such as quenchers, are introduced into polynucleotide used in the methods described herein, such as FRET probes, via modified nucleotides. A “modified nucleotide” refers to a nucleotide that has been chemically modified, but still functions as a nucleotide. In some embodiments, the modified nucleotide has a chemical moiety, such as a dye or quencher, covalently attached, and can be introduced into a polynucleotide, for example, by way of solid phase synthesis of the polynucleotide. In other embodiments, the modified nucleotide includes one or more reactive groups that can react with a dye or quencher before, during, or after incorporation of the modified nucleotide into the nucleic acid. In specific embodiments, the modified nucleotide is an amine-modified nucleotide, i.e., a nucleotide that has been modified to have a reactive amine group. In some embodiments, the modified nucleotide comprises a modified base moiety, such as uridine, adenosine, guanosine, and/or cytosine. In specific embodiments, the amine-modified nucleotide is selected from 5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and 8-[(6-amino)butyl]-amino-ATP; N6-(4-amino)butyl-ATP, N6-(6-amino)butyl-ATP, N4-[2,2-oxy-bis-(ethylamine)]-CTP; N6-(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP; 5-propargylamino-CTP, 5-propargylamino-UTP. In some embodiments, nucleotides with different nucleobase moieties are similarly modified, for example, 5-(3-aminoallyl)-GTP instead of 5-(3-aminoallyl)-UTP. Many amine modified nucleotides are commercially available from, e.g., Applied Biosystems, Sigma, Jena Bioscience and TriLink.
Exemplary detectable moieties also include, but are not limited to, members of binding pairs. In some such embodiments, a first member of a binding pair is linked to a polynucleotide. The second member of the binding pair is linked to a detectable label, such as a fluorescent label. When the polynucleotide linked to the first member of the binding pair is incubated with the second member of the binding pair linked to the detectable label, the first and second members of the binding pair associate and the polynucleotide can be detected. Exemplary binding pairs include, but are not limited to, biotin and streptavidin, antibodies and antigens, etc.
In some embodiments, multiple target RNAs are detected in a single multiplex reaction. In some such embodiments, each probe that is targeted to a unique cDNA is spectrally distinguishable when released from the probe. Thus, each target RNA is detected by a unique fluorescence signal.
One skilled in the art can select a suitable detection method for a selected assay, e.g., a real-time RT-PCR assay. The selected detection method need not be a method described above, and may be any method.
4.2. Exemplary Compositions and Kits
In another aspect, compositions are provided. In some embodiments, compositions are provided for use in the methods described herein.
In some embodiments, a composition comprises at least one polynucleotide. In some embodiments, a composition comprises at least one primer. In some embodiments, a composition comprises at least one probe. In some embodiments, a composition comprises at least one primer and at least one probe.
In some embodiments, compositions are provided that comprise at least one target RNA-specific primer. The term “target RNA-specific primer” encompasses primers that have a region of contiguous nucleotides having a sequence that is (i) identically present in one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897, or (ii) complementary to the sequence of a region of contiguous nucleotides found in one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897.
In some embodiments, compositions are provided that comprise at least one target RNA-specific probe. The term “target RNA-specific probe” encompasses probes that have a region of contiguous nucleotides having a sequence that is (i) identically present in one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897, or (ii) complementary to the sequence of a region of contiguous nucleotides found in one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897.
In some embodiments, target RNA-specific primers and probes comprise deoxyribonucleotides. In other embodiments, target RNA-specific primers and probes comprise at least one nucleotide analog. Nonlimiting exemplary nucleotide analogs include, but are not limited to, analogs described herein, including LNA analogs and peptide nucleic acid (PNA) analogs. In some embodiments, target RNA-specific primers and probes comprise at least one nucleotide analog which increases the hybridization binding energy (e.g., an affinity-enhancing nucleotide analog, discussed above). In some embodiments, a target RNA-specific primer or probe in the compositions described herein binds to one target RNA in the sample. In some embodiments, a single primer or probe binds to multiple target RNAs, such as multiple isomirs.
In some embodiments, more than one primer or probe specific for a single target RNA is present in the compositions, the primers or probes capable of binding to overlapping or spatially separated regions of the target RNA.
It will be understood, even if not explicitly stated hereinafter, that in some embodiments in which the compositions described herein are designed to hybridize to cDNAs reverse transcribed from target RNAs, the composition comprises at least one target RNA-specific primer or probe (or region thereof) having a sequence that is identically present in a target RNA (or region thereof).
In some embodiments, a target RNA is capable of specifically hybridizing to at least one probe comprising a sequence selected from SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 and 86. In some embodiments, a target RNA is capable of specifically hybridizing to at least one nucleic acid probe comprising a sequence selected from SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 and 79. In some embodiments, a target RNA is capable of specifically hybridizing to at least one nucleic acid probe comprising a sequence selected from SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 and 84. In some embodiments, a target RNA is capable of specifically hybridizing to at least one nucleic acid probe comprising a sequence selected from SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, and 78. In some embodiments, a target RNA is capable of specifically hybridizing to at least one nucleic acid probe comprising a sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 and 85. In some embodiments, a target RNA is capable of specifically hybridizing to at least one nucleic acid probe comprising a sequence selected from SEQ ID NOs: 9, 50, 51, 70, 72 and 75. In some embodiments, a target RNA is capable of specifically hybridizing to at least one probe comprising a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, a target RNA comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, a target RNA comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, and 648. In some embodiments, a target RNA comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, and 632. In some embodiments, a target RNA comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 226 to 289, 565 to 604, and 863 to 868. In some embodiments, a target RNA comprises a sequence that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, a target RNA, in its mature form, comprises fewer than 30 nucleotides. In some embodiments, a target RNA is a microRNA.
In some embodiments, the composition comprises a plurality of target RNA-specific primers and/or probes for each of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 target RNAs, the target RNAs comprising a region of contiguous nucleotides having a sequence that is identically present in one of SEQ ID NOs: 87 to 177, 948, 400 to 564, 949, 708 to 862, and 898 to 932. In some embodiments, the plurality includes a target RNA-specific primer and/or probe specific for each of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 target RNAs, the target RNAs comprising a region of contiguous nucleotides having a sequence that is identically present in one of SEQ ID NOs: 87 to 177, 948, 400 to 564, 949, 708 to 862, and 898 to 932. In some embodiments, the plurality includes a target RNA-specific primer and/or probe specific for each of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100 target RNAs comprising a region of contiguous nucleotides having a sequence that is identically present in one of SEQ ID NOs: 87 to 177, 948, 400 to 564, 949, 708 to 862, and 898 to 932. It will be understood that, in some embodiments, target RNAs described herein comprise a sequence identically present in a sequence set forth in Table 3, 13, 15, or 17, except that thymine (T) bases in the sequences shown in Table 3, 13, 15, or 17 are replaced by uracil (U) bases in the target RNAs.
In some embodiments, a composition is an aqueous composition. In some embodiments, the aqueous composition comprises a buffering component, such as phosphate, tris, HEPES, etc., and/or additional components, as discussed below. In some embodiments, a composition is dry, for example, lyophilized, and suitable for reconstitution by addition of fluid. A dry composition may include a buffering component and/or additional components.
In some embodiments, a composition comprises one or more additional components. Additional components include, but are not limited to, salts, such as NaCl, KCl, and MgCl2; polymerases, including thermostable polymerases; dNTPs; RNase inhibitors; bovine serum albumin (BSA) and the like; reducing agents, such as β-mercaptoethanol; EDTA and the like; etc. One skilled in the art can select suitable composition components depending on the intended use of the composition.
In some embodiments, an addressable microarray component is provided that comprises target RNA-specific probes attached to a substrate.
Microarrays for use in the methods described herein comprise a solid substrate onto which the probes are covalently or non-covalently attached. In some embodiments, probes capable of hybridizing to one or more target RNAs or cDNAs are attached to the substrate at a defined location (“addressable array”). Probes can be attached to the substrate in a wide variety of ways, as will be appreciated by those in the art. In some embodiments, the probes are synthesized first and subsequently attached to the substrate. In other embodiments, the probes are synthesized on the substrate. In some embodiments, probes are synthesized on the substrate surface using techniques such as photopolymerization and photolithography.
In some embodiments, the solid substrate is a material that is modified to contain discrete individual sites appropriate for the attachment or association of the probes and is amenable to at least one detection method. Representative examples of substrates include glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses and plastics. In some embodiments, the substrates allow optical detection without appreciably fluorescing.
In some embodiments, the substrate is planar. In other embodiments, probes are placed on the inside surface of a tube, such as for flow-through sample analysis to minimize sample volume. In other embodiments, probes can be in the wells of multi-well plates. In still other embodiments, probes can be attached to an addressable microbead array. In yet other embodiments, the probes can be attached to a flexible substrate, such as a flexible foam, including closed cell foams made of particular plastics.
The substrate and the probe can each be derivatized with functional groups for subsequent attachment of the two. For example, in some embodiments, the substrate is derivatized with one or more chemical functional groups including, but not limited to, amino groups, carboxyl groups, oxo groups and thiol groups. In some embodiments, probes are attached directly to the substrate through one or more functional groups. In some embodiments, probes are attached to the substrate indirectly through a linker (i.e., a region of contiguous nucleotides that space the probe regions involved in hybridization and detection away from the substrate surface). In some embodiments, probes are attached to the solid support through the 5′ terminus. In other embodiments, probes are attached through the 3′ terminus. In still other embodiments, probes are attached to the substrate through an internal nucleotide. In some embodiments the probe is attached to the solid support non-covalently, e.g., via a biotin-streptavidin interaction, wherein the probe biotinylated and the substrate surface is covalently coated with streptavidin.
In some embodiments, the compositions comprise a microarray having probes attached to a substrate, wherein at least one of the probes (or a region thereof) comprises a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897. In some embodiments, at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, or at least 100 of the probes comprise a sequence that is identically present in, or complementary to a region of one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897. In some embodiments, the microarray comprises at least one target RNA-specific probe comprising a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897 and at least one target RNA-specific probe comprising a sequence that is identically present in, or complementary to a region of a target RNA of the human miRNome. In some embodiments, the microarray comprises each target RNA-specific probe at only one location on the microarray. In some embodiments, the microarray comprises at least one target RNA-specific probe at multiple locations on the microarray.
As used herein, the terms “complementary” or “partially complementary” to a target RNA (or target region thereof), and the percentage of “complementarity” of the probe sequence to that of the target RNA sequence is the percentage “identity” to the reverse complement of the sequence of the target RNA. In determining the degree of “complementarity” between probes used in the compositions described herein (or regions thereof) and a target RNA, such as those disclosed herein, the degree of “complementarity” is expressed as the percentage identity between the sequence of the probe (or region thereof) and the reverse complement of the sequence of the target RNA that best aligns therewith. The percentage is calculated by counting the number of aligned bases that are identical as between the 2 sequences, dividing by the total number of contiguous nucleotides in the probe, and multiplying by 100.
In some embodiments, the microarray comprises at least one probe having a region with a sequence that is fully complementary to a target region of a target RNA. In other embodiments, the microarray comprises at least one probe having a region with a sequence that comprises one or more base mismatches when compared to the sequence of the best-aligned target region of a target RNA.
As noted above, a “region” of a probe or target RNA, as used herein, may comprise or consist of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or more contiguous nucleotides from a particular SEQ ID NO or the complement thereof. In some embodiments, the region is of the same length as the probe or the target RNA. In other embodiments, the region is shorter than the length of the probe or the target RNA.
In some embodiments, the microarray comprises at least one probe having a region of at least 10, at least 11, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 68, 196 to 399, 950, 565 to 707, or 863 to 897.
In some embodiments, the microarray comprises at least one probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 or 86. In some embodiments, the microarray comprises at least one, at least two, at least three, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or at lest 40 probes that each comprise a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 and 86. In some embodiments, the microarray further comprises additional probes that do not have a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 or 86.
In some embodiments, the microarray comprises at least one probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 or 79. In some embodiments, the microarray comprises at least one, at least two, at least three, at least five, at least eight, at least 10, at least 12, or at least 15 probes that each comprise a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 and 79. In some embodiments, the microarray further comprises additional probes that do not have a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 and 79.
In some embodiments, the microarray comprises at least one probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 or 84. In some embodiments, the microarray comprises at least one, at least two, at least three, at least five, at least eight, at least 10, at least 12, or at least 15 probes that each comprise a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 or 84. In some embodiments, the microarray further comprises additional probes that do not have a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 or 84.
In some embodiments, the microarray comprises at least one probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, or 78. In some embodiments, the microarray comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight probes that each comprise a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, or 78. In some embodiments, the microarray further comprises additional probes that do not have a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, or 78.
In some embodiments, the microarray comprises at least one probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85. In some embodiments, the microarray comprises at least one, at least two, at least three, at least four, at least five, at least ten, at least 15, at least 20, at least 25, or at least 30 probes that each comprise a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85. In some embodiments, the microarray further comprises additional probes that do not have a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85.
In some embodiments, the microarray comprises at least one probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 9, 50, 51, 70, 72 or 75. In some embodiments, the microarray comprises at least one, at least two, at least three, at least four, at least five, or at least six probes that each comprise a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 9, 50, 51, 70, 72 or 75. In some embodiments, the microarray further comprises additional probes that do not have a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 9, 50, 51, 70, 72 or 75.
In some embodiments, the microarray comprises at least one probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, or 648. In some embodiments, the microarray comprises at least two, at least five, at least 10, at least 15, at least 20, at least 25, or at least 30 probes that each comprise a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, or 648. In some embodiments, the microarray further comprises additional probes that do not have a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, or 648.
In some embodiments, the microarray comprises at least one probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632. In some embodiments, the microarray comprises at least two, at least five, at least 10, at least 15, or at least 20 probes that each comprise a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632. In some embodiments, the microarray further comprises additional probes that do not have a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632.
In some embodiments, the microarray comprises at least one probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868. In some embodiments, the microarray comprises at least two, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, or at least 70 probes that each comprise a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868. In some embodiments, the microarray further comprises additional probes that do not have a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868.
In some embodiments, the microarrays comprise probes having a region with a sequence that is complementary to target RNAs that comprise a substantial portion of the human miRNome (i.e., the publicly known microRNAs that have been accessioned by others into miRBase (microma.sanger.ac.uk/ at the time the microarray is fabricated), such as at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the human miRNome. In some embodiments, the microarrays comprise probes that have a region with a sequence that is identically present in target RNAs that comprise a substantial portion of the human miRNome, such as at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the human miRNome.
In some embodiments, components are provided that comprise probes attached to microbeads, such as those sold by Luminex, each of which is internally dyed with red and infrared fluorophores at different intensities to create a unique signal for each bead. In some embodiments, the compositions useful for carrying out the methods described herein include a plurality of microbeads, each with a unique spectral signature. Each uniquely labeled microbead is attached to a unique target RNA-specific probe such that the unique spectral signature from the dyes in the bead is associated with a particular probe sequence. Nonlimiting exemplary probe sequences include SEQ ID NOs: 1 to 86. Nonlimiting exemplary probe sequences also include probes comprising a region that is identically present in, or complementary to, a sequence selected from SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, a probe sequence comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides that are identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897.
In some embodiments, a uniquely labeled microbead has attached thereto a probe having a region with a sequence that is identically present in, or complementary to a region of one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897. In other embodiments, the uniquely labeled microbead has attached thereto a probe having a region with a sequence that comprises one or more base mismatches when compared to the most similar sequence selected from SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897, and sequences complementary to SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, and 863 to 897.
In some embodiments, a composition is provided that comprises a plurality of uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe having a region of at least 10, at least 11, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897.
In some embodiments, the compositions comprise a plurality of uniquely labeled microbeads, at least one of which has attached thereto a target RNA-specific probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 or 86. In some embodiments, the compositions comprise at least two, at least three, at least five, at least 8, at least ten, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 uniquely labeled microbeads that each have attached thereto a unique target RNA-specific probe having a region with a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 or 86. In some embodiments, the composition comprises at least one uniquely labeled microbead having attached thereto a target RNA-specific probe having a region with a sequence that is not present in, or complementary to a region of, any of SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 or 86
In some embodiments, the compositions comprise a plurality of uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 or 79. In some embodiments, the compositions comprise at least two, at least three, at least five, at least eight, at least 10, at least 12, at least 15, or at least 18 uniquely labeled microbeads that each have attached thereto a unique target RNA-specific probe having a region with a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 or 79. In some embodiments, the composition comprises at least one uniquely labeled microbead having attached thereto a target RNA-specific probe having a region with a sequence that is not present in, or complementary to a region of, any of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 or 79.
In some embodiments, the compositions comprise a plurality of uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 or 84. In some embodiments, the compositions comprise at least two, at least three, at least five, at least eight, at least 10, at least 12, at least 15, or at least 18 uniquely labeled microbeads that each have attached thereto a unique target RNA-specific probe having a region with a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 or 84. In some embodiments, the composition comprises at least one uniquely labeled microbead having attached thereto a target RNA-specific probe having a region with a sequence that is not present in, or complementary to a region of, any of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 or 84.
In some embodiments, the compositions comprise a plurality of uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, or 78. In some embodiments, the compositions comprise at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight uniquely labeled microbeads that each have attached thereto a unique target RNA-specific probe having a region with a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, or 78. In some embodiments, the composition comprises at least one uniquely labeled microbead having attached thereto a target RNA-specific probe having a region with a sequence that is not present in, or complementary to a region of, any of SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, or 78.
In some embodiments, the compositions comprise a plurality of uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85. In some embodiments, the compositions comprise at least two, at least three, at least five, at least 8, at least ten, at least 15, at least 20, at least 25, at least 30, or at least 35 uniquely labeled microbeads that each have attached thereto a unique target RNA-specific probe having a region with a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85. In some embodiments, the composition comprises at least one uniquely labeled microbead having attached thereto a target RNA-specific probe having a region with a sequence that is not present in, or complementary to a region of, any of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85.
In some embodiments, the compositions comprise a plurality of uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 9, 50, 51, 70, 72 or 75. In some embodiments, the compositions comprise at least two, at least three, at least four, at least five, or at least six uniquely labeled microbeads that each have attached thereto a unique target RNA-specific probe having a region with a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 9, 50, 51, 70, 72 or 75. In some embodiments, the composition comprises at least one uniquely labeled microbead having attached thereto a target RNA-specific probe having a region with a sequence that is not present in, or complementary to a region of, any of SEQ ID NOs: 9, 50, 51, 70, 72 or 75.
In some embodiments, the compositions comprise a plurality of uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe having a region with a sequence that is identically present in, or complementary to a region of one of SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, or 648. In some embodiments, the compositions comprise at least two, at least five, at least 10, at least 15, at least 20, at least 25, or at least 30 uniquely labeled microbeads that each have attached thereto a unique target RNA-specific probe having a region with a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, or 648. In some embodiments, the composition comprises at least one uniquely labeled microbead having attached thereto a target RNA-specific probe having a region with a sequence that is not present in, or complementary to a region of, any of SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, or 648.
In some embodiments, the compositions comprise a plurality of uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632. In some embodiments, the compositions comprise at least two, at least five, at least 10, at least 15, or at least 20 uniquely labeled microbeads that each have attached thereto a unique target RNA-specific probe having a region with a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632. In some embodiments, the composition comprises at least one uniquely labeled microbead having attached thereto a target RNA-specific probe having a region with a sequence that is not present in, or complementary to a region of, any of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632.
In some embodiments, the compositions comprise a plurality of uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868. In some embodiments, the compositions comprise at least two, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, or at least 70 uniquely labeled microbeads that each have attached thereto a unique target RNA-specific probe having a region with a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868. In some embodiments, the composition comprises at least one uniquely labeled microbead having attached thereto a target RNA-specific probe having a region with a sequence that is not present in, or complementary to a region of, any of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868.
In some embodiments, the compositions comprise a plurality of uniquely labeled microbeads, wherein the plurality comprises at least one microbead having attached thereto a probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897. In some embodiments, the plurality comprises at least two, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 75, or at least 100 microbeads each of which having attached thereto a probe having a region with a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897. In some embodiments, a composition comprises at least one uniquely labeled microbead having attached thereto a target RNA-specific probe having a region with a sequence that is not present in, or complementary to a region of, any of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897.
In some embodiments, the compositions comprise a plurality of uniquely labeled microbeads, at least one of which has attached thereto a probe having a region with a sequence that identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897 and at least a second bead that has attached thereto a probe having a region with a sequence that is identically present in, or complementary to a region of, a target RNA from the human miRNome.
In some embodiments, the compositions comprise a plurality of uniquely labeled microbeads, each of which has attached thereto a unique probe having a region that is complementary to target RNAs that comprise a substantial portion of the human miRNome, such as at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the human miRNome. In some embodiments, the compositions comprise a plurality of uniquely labeled microbeads having attached thereto a unique probe having a region with a sequence that is identically present in target RNAs that comprise a substantial portion of the human miRNome, such as at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the human miRNome.
In some embodiments, compositions are provided that comprise at least one polynucleotide for detecting at least one target RNA. In some embodiments, the polynucleotide is used as a primer for a reverse transcriptase reaction. In some embodiments, the polynucleotide is used as a primer for amplification. In some embodiments, the polynucleotide is used as a primer for RT-PCR. In some embodiments, the polynucleotide is used as a probe for detecting at least one target RNA. In some embodiments, the polynucleotide is detectably labeled. In some embodiments, the polynucleotide is a FRET probe. In some embodiments, the polynucleotide is a TaqMan® probe, a Molecular Beacon, or a Scorpion probe.
In some embodiments, a composition comprises at least one FRET probe having a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897. In some embodiments, a composition comprises at least two, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 75, or at least 100 FRET probes, each of which has a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897. In some embodiments, a FRET probe is labeled with a donor/acceptor pair such that when the probe is digested during the PCR reaction, it produces a unique fluorescence emission that is associated with a specific target RNA. In some embodiments, when a composition comprises multiple FRET probes, each probe is labeled with a different donor/acceptor pair such that when the probe is digested during the PCR reaction, each one produces a unique fluorescence emission that is associated with a specific probe sequence and/or target RNA. In some embodiments, the sequence of the FRET probe is complementary to a target region of a target RNA. In other embodiments, the FRET probe has a sequence that comprises one or more base mismatches when compared to the sequence of the best-aligned target region of a target RNA.
In some embodiments, a composition comprises a FRET probe consisting of at least 8, at least 9, at least 10, at least 11, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides, wherein at least a portion of the sequence is identically present in, or complementary to a region of one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897. In some embodiments, at least 8, at least 9, at least 10, at least 11, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides of the FRET probe are identically present in, or complementary to a region of one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897. In some embodiments, the FRET probe has a sequence with one, two or three base mismatches when compared to the sequence or complement of one of SEQ ID NOs: 1 to 86, 196 to 399, 950, 565 to 707, or 863 to 897.
In some embodiments, the compositions comprise at least one target RNA-specific FRET probe comprising a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 or 86. In some embodiments, the compositions comprise at least two, at least three, at least five, at least 8, at least ten, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 uniquely labeled target RNA-specific FRET probes, each comprising a sequence that is identically present in, or complementary to a region of, a different one of 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 or 86.
In some embodiments, the compositions comprise at least one target RNA-specific FRET probe comprising a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 or 79. In some embodiments, the compositions comprise at least two, at least three, at least five, at least eight, at least 10, at least 12, at least 15, or at least 18 uniquely labeled target RNA-specific FRET probes, each of which comprises a sequence that is identically present in, or complementary to a region of a different one of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 or 79.
In some embodiments, the compositions comprise at least one target RNA-specific FRET probe comprising a sequence that is identically present in, or complementary to a region of one of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 or 84. In some embodiments, the compositions comprise at least two, at least three, at least five, at least eight, at least 10, at least 12, at least 15, or at least 18 uniquely labeled target RNA-specific FRET probes, each of which comprises a sequence that is identically present in, or complementary to a region of a different one of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 or 84.
In some embodiments, the compositions comprise at least one target RNA-specific FRET probe comprising a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, or 78. In some embodiments, the compositions comprise at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight uniquely labeled target RNA-specific FRET probes, each of which comprises a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, or 78.
In some embodiments, the compositions comprise at least one target RNA-specific FRET probe comprising a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85. In some embodiments, the compositions comprise at least two, at least three, at least five, at least 8, at least ten, at least 15, at least 20, at least 25, at least 30, or at least 35 uniquely labeled target RNA-specific FRET probes, each of which comprises a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85.
In some embodiments, the compositions comprise at least one target RNA-specific FRET probe comprising a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 9, 50, 51, 70, 72 or 75. In some embodiments, the compositions comprise at least two, at least three, at least four, at least five, or at least six uniquely labeled target RNA-specific FRET probes, each of which comprises a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 9, 50, 51, 70, 72 or 75.
In some embodiments, the compositions comprise at least one target RNA-specific FRET probe comprising a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, or 648. In some embodiments, the compositions comprise at least two, at least five, at least 10, at least 15, at least 20, at least 25, or at least 30 uniquely labeled target RNA-specific FRET probes, each of which comprises a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, or 648.
In some embodiments, the compositions comprise at least one target RNA-specific FRET probe comprising a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632. In some embodiments, the compositions comprise at least two, at least five, at least 10, at least 15, or at least 20 uniquely labeled target RNA-specific FRET probes, each of which comprises a sequence that is identically present in, or complementary to a region of, a different one of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632.
In some embodiments, the compositions comprise at least one target RNA-specific FRET probe comprising a sequence that is identically present in, or complementary to a region of, one of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868. In some embodiments, the compositions comprise at least two, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, or at least 70 uniquely labeled target RNA-specific FRET probes, each of which comprises a sequence that is identically present in, or complementary to a region of a different one of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868.
In some embodiments, a kit comprises a polynucleotide discussed above. In some embodiments, a kit comprises at least one primer and/or probe discussed above. In some embodiments, a kit comprises at least one polymerase, such as a thermostable polymerase. In some embodiments, a kit comprises dNTPs. In some embodiments, kits for use in the real time RT-PCR methods described herein comprise one or more target RNA-specific FRET probes and/or one or more primers for reverse transcription of target RNAs and/or one or more primers for amplification of target RNAs or cDNAs reverse transcribed therefrom.
In some embodiments, one or more of the primers and/or probes is “linear”. A “linear” primer refers to a polynucleotide that is a single stranded molecule, and typically does not comprise a short region of, for example, at least 3, 4 or 5 contiguous nucleotides, which are complementary to another region within the same polynucleotide such that the primer forms an internal duplex. In some embodiments, the primers for use in reverse transcription comprise a region of at least 4, at least 5, at least 6, at least 7 or more contiguous nucleotides at the 3′-end that has a sequence that is complementary to region of at least 4, at least 5, at least 6, at least 7 or more contiguous nucleotides at the 5′-end of a target RNA.
In some embodiments, a kit comprises one or more pairs of linear primers (a “forward primer” and a “reverse primer”) for amplification of a cDNA reverse transcribed from a target RNA. Accordingly, in some embodiments, a first primer comprises a region of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides having a sequence that is identical to the sequence of a region of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides at the 5′-end of a target RNA. Furthermore, in some embodiments, a second primer comprises a region of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides having a sequence that is complementary to the sequence of a region of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous nucleotides at the 3′-end of a target RNA. In some embodiments, the kit comprises at least a first set of primers for amplification of a cDNA that is reverse transcribed from a target RNA capable of specifically hybridizing to a nucleic acid comprising a sequence identically present in one of SEQ ID NOs: 1 to 86 and/or a cDNA that is reverse transcribed from a target RNA that comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897.
In some embodiments, the kit comprises at least two, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 75, or at least 100 sets of primers, each of which is for amplification of a cDNA that is reverse transcribed from a different target RNA capable of specifically hybridizing to a sequence selected from SEQ ID NOs: 1 to 86 and/or a cDNA that is reverse transcribed from a target RNA that comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707 and 863 to 897. In some embodiments, the kit comprises at least one set of primers that is capable of amplifying more than one cDNA reverse transcribed from a target RNA in a sample.
In some embodiments, probes and/or primers for use in the compositions described herein comprise deoxyribonucleotides. In some embodiments, probes and/or primers for use in the compositions described herein comprise deoxyribonucleotides and one or more nucleotide analogs, such as LNA analogs or other duplex-stabilizing nucleotide analogs described above. In some embodiments, probes and/or primers for use in the compositions described herein comprise all nucleotide analogs. In some embodiments, the probes and/or primers comprise one or more duplex-stabilizing nucleotide analogs, such as LNA analogs, in the region of complementarity.
In some embodiments, the compositions described herein also comprise probes, and in the case of RT-PCR, primers, that are specific to one or more housekeeping genes for use in normalizing the quantities of target RNAs. Such probes (and primers) include those that are specific for one or more products of housekeeping genes selected from U6 snRNA, RNU44, RNU48, U47, 7SL scRNA, U1 snRNA, 5.8S rRNA, and U87 scaRNA.
In some embodiments, the kits for use in real time RT-PCR methods described herein further comprise reagents for use in the reverse transcription and amplification reactions. In some embodiments, the kits comprise enzymes such as reverse transcriptase, and a heat stable DNA polymerase, such as Taq polymerase. In some embodiments, the kits further comprise deoxyribonucleotide triphosphates (dNTPs) for use in reverse transcription and amplification. In further embodiments, the kits comprise buffers optimized for specific hybridization of the probes and primers.
4.2.1. Exemplary Normalization of RNA Levels
In some embodiments, quantitation of target RNA expression levels requires assumptions to be made about the total RNA per cell and the extent of sample loss during sample preparation. In order to correct for differences between different samples or between samples that are prepared under different conditions, the quantities of target RNAs in some embodiments are normalized to the expression of at least one endogenous housekeeping gene.
Appropriate genes for use as reference genes in the methods described herein include those as to which the quantity of the product does not vary between normal samples and samples from sepsis patients, or between different cell lines or under different growth and sample preparation conditions. In some embodiments, endogenous housekeeping genes useful as normalization controls in the methods described herein include, but are not limited to, U6 snRNA, RNU44, RNU48, U47, 7SL scRNA, U1 snRNA, 5.8S rRNA, and U87 scaRNA. In typical embodiments, the at least one endogenous housekeeping gene for use in normalizing the measured quantity of microRNAs is selected from U6 snRNA, RNU44, RNU48, U47, 7SL scRNA, U1 snRNA, 5.8S rRNA, and U87 scaRNA. In some embodiments, one housekeeping gene is used for normalization. In some embodiments, more than one housekeeping gene is used for normalization.
4.2.2. Exemplary Qualitative Methods
In some embodiments, methods comprise detecting a qualitative change in a target RNA profile generated from a human sample as compared to a normal target RNA profile (in some exemplary embodiments, a target RNA profile of a control sample). Some qualitative changes in the expression profile are indicative of the presence of sepsis in a sample from a subject. The term “target RNA profile” refers to a set of data regarding the concurrent expression of a plurality of target RNAs in the same sample.
In some embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 of the target RNAs of the plurality of target RNAs are capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 and 86. In some embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 15, or at least 18 of the target RNAs of the plurality of target RNAs is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 and 79. In some embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 15, or at least 18 of the target RNAs of the plurality of target RNAs is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 and 84. In some embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight of the target RNAs of the plurality of target RNAs is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, and 78. In some embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, or at least 35 of the target RNAs of the plurality of target RNAs is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 and 85. In some embodiments, at least one, at least two, at least three, at least four, at least five, or at least six of the target RNAs of the plurality of target RNAs is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 9, 50, 51, 70, 72 and 75.
In some embodiments, at least one, at least two, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, or at least 75 of the plurality of target RNAs is capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, at least one, at least two, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100 of the plurality of target RNAs comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897. In some embodiments, at least one, at least two, at least five, at least 10, at least 15, at least 20, or at least 25 of the plurality of target RNAs comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, and 648. In some embodiments, at least one, at least two, at least five, at least 10, at least 15, at least 20, or at least 25 of the plurality of target RNAs comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, and 632. In some embodiments, at least one, at least two, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, or at least 70 of the plurality of target RNAs comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 226 to 289, 565 to 604, and 863 to 868. In some embodiments, at least one, at least two, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, or at least 75 of the plurality of target RNAs comprises a sequence that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, a target RNA, in its mature form, comprises fewer than 30 nucleotides. In some embodiments, a target RNA is a microRNA.
Qualitative expression data for use in preparing target RNA expression profiles is obtained using any suitable analytical method, including the analytical methods presented herein.
In some embodiments, for example, concurrent expression data are obtained using, e.g., a microarray, as described above. Thus, in addition to use for quantitative expression level assays of specific target RNAs as described above, a microarray comprising probes having sequences that are complementary to a substantial portion of the miRNome may be employed to cam/out target RNA gene expression profiling, for analysis of target RNA expression patterns.
In some embodiments, distinct target RNA signatures are associated with established markers for sepsis. In some embodiments, distinct target RNA signatures are associated with established markers for sepsis caused by bacterial infection, such as for sepsis caused by gram-positive bacterial infection, sepsis caused by gram-negative bacterial infection or sepsis caused by mycobacterial infection. In some embodiments, distinct target RNA signatures are associated with established markers for sepsis caused by viral infection. In some embodiments, distinct target RNA signatures are associated with established markers for sepsis caused by multiple infection, such as by co-infection with bacteria and viruses, or by co-infection with more than one viral or more than one bacterial strain. In some embodiments, distinct target RNA signatures are associated directly with the level of severity of the sepsis.
According to the expression profiling method, in some embodiments, total RNA from a sample from a subject suspected of having sepsis is quantitatively reverse transcribed to provide a set of labeled oligonucleotides complementary to the RNA in the sample. The oligonucleotides are then hybridized to a microarray comprising target RNA-specific probes to provide a hybridization profile for the sample. The result is a hybridization profile for the sample representing the expression pattern of target RNAs in the sample. The hybridization profile comprises the signal from the binding of the oligonucleotides reverse transcribed from the sample to the target RNA-specific probes in the microarray. In some embodiments, the profile is recorded as the presence or absence of binding (signal vs. zero signal). In some embodiments, the profile recorded includes the intensity of the signal from each hybridization. The profile is compared to the hybridization profile generated from a normal, i.e., nonseptic sample, or in some embodiments, a control sample. An alteration in the signal is indicative of the presence of sepsis in the subject.
4.3. Exemplary Additional Target RNAs
In some embodiments, in combination with detecting one or more target RNAs that are capable of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs:1 to 86 and/or detecting one or more target RNAs comprising at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 950, 565 to 707, and 863 to 897 and/or detecting one or more target RNAs that comprise a sequence that is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs:1 to 86, methods herein further comprise detecting the level(s) of expression of at least one other marker associated with sepsis.
In some embodiments, the methods described herein further comprise detecting altered expression of sepsis-associated small RNAs with non-canonical hairpins.
In alternative embodiments, the methods described herein further comprise detecting chromosomal codependents, i.e., target RNAs clustered near each other in the human genome which tend to be regulated together. Accordingly, in further embodiments, the methods comprise detecting the expression of one or more target microRNAs, each situated within the chromosome no more than 50,000 bp from the chromosomal location of the pre-microRNA sequences in Table 2.
The following examples are for illustration purposes only, and are not meant to be limiting in any way.
5. EXAMPLES
5.1 Example 1
MicroRNAs from Monocytes
Using microarray analysis, distinct microRNAs were demonstrated to be overexpressed in monocytes in response to stimulation with pathogen mimics (agonists).
Cell Lines
Total RNA was prepared from the monocyte cell line THP-1 (ATCC No. TIB-202), which is an acute monocytic leukemia cell line of human peripheral blood origin.
Stimulation of Monocytes
Both THP-1 cells and pooled human monocytes from healthy donors were stimulated with different Toll-Like Receptors (TLR) agonists set forth in Table 5 below.










TABLE 5







TLR Stimulated
Agonist









TLR2a
1 μg/ml Pam3CSK4 (Invitrogen)



TLR2b
10 μg/ml peptidoglycan (Invitrogen)



TLR3
25 μg/ml poly(I:C) (Invitrogen)



TLR4a
10 μg/ml ultrapure LPS (from E. coli strain




K12, Invitrogen)



TLR4b
1 μg/ml LPS (E. coli 055:B5, Sigma)



TLR5
100 ng/ml Flagellin (S. typhimurium,




Invitrogen)



TLR7
5 μg/ml imiquimod-R837(Invitrogen)



TLR9
5 μM CpG oligonucleotide (Invitrogen)




50 ng/ml phorbol myristyl acetate in




combination with 1 uM ionomycin




RPMI medium alone










THP-1 cells were obtained from American Type Culture Collection (Manassas, Va.). THP-1 cells were grown to a concentration of 2.5 million per milliliter in RPMI medium 1640 supplemented with 10% FBS, 1× nonessential amino acids, 100 units/ml penicillin, 100 units/ml streptomycin, and 2 mM glutamine in a humidified incubator containing 5% CO2 at 37° C. Human monocytes were isolated from whole blood of healthy donors (St Guy's hospital, UK), using CD14 positive magnetic microbead positive selection (according to manufacturer's protocol, Miltenyi Biotec). Isolated monocytes were then cultured with RPMI 1640 medium to a concentration of 2.5 million per milliliter in 6-well plates and cultured in a humidified incubator containing 5% CO2 at 37° C.
To analyze miRNA expression, cells were treated for 8 h or 24 h with the stimuli shown above in Table 5. Concentrations were chosen according to manufactory recommendations and publication: Taganov, K. et al. PNAS, 103(33), p. 12481-6.
Total RNA was isolated by using standard TRIzol® protocol (Invitrogen). Cells from two confluent 75 cm2 flasks were harvested (=approx 107 cells). Total RNA was prepared using TRIzol® Reagent, Invitrogen (Carlsbad, Calif.) according to the manufacturer's protocol. All RNA samples were diluted in RNase-free water and stored in −80° C. (−112° F.).
RNA quality was assessed by calculating OD 260/280 ratios. The quality of all RNA samples was high as assessed using an Agilent Bioanalyser 2100, as exemplified by the electropherogram shown in FIG. 1 obtained for total RNA from human monocyte cell line THP-1 after stimulation for 8 h with agonist Pam3CSK4. Similar electropherograms were obtained for total RNA from the other cell samples as well.
MicroRNA Purification
MicroRNA purification was performed using a Flash PAGE Fractionator (Ambion). The Ambion gel purification protocol enriches for small RNAs less than 40 nucleotides (nt) long, including microRNAs. Briefly, a total RNA sample was loaded onto a pre-cast gel using the Flash PAGE Fractionator. The total RNA fraction smaller than 40 nt (the “microRNA fraction”) was recovered after gel migration and resuspended into nuclease free water.
Microarray Analysis
Probe Design and Spotting
The oligonucleotide probes used for microarray preparation had the configuration 5′-NH2—(C)6-(spacer)-(oligomer probe sequence)-3′. The 5′-amino group allowed chemical bonding onto the array support. Each also included an identical spacer sequence of 15 nt, as shown below, to prevent non-specific interactions of the oligonucleotide probes with the array support:







(SEQ ID NO: 933)


5′AminoC6-TTGTAATACGACTCA-Oligo probe sequence.






Probe sequences given in Table 2 omit the linker.

The probes were synthesized according to standard protocols by Eurofins MWG Operon (Ebersberg, Germany). Nexterion (Schott) microarray glass slides were used as the solid support for the microarray.
The oligonucleotide probe concentration used for the spotting was 25 μmol. The probes were spotted in duplicate using the Nexterion spotting buffer provided with the array glass support by Schott with 1 SDS (sodium dodecyl sulfate) added to allow larger spat sizes (e.g., 100-150 microns compared to 70-100 microns without SDS). The spotter used was the QArray mini (Genetix) equipped with Stealth SMP3 pins (Telechem). After deposition of one series of spots, the spotting needle was washed 5 times with 60 mM NaOH before spotting the next series of probes. Each slide is designed with 32 blocks of spotted probes, with each block being a 20×20 square of spotted probes. Each probe was spotted in duplicate. Spotted glass slides were stored at 4° C. until use.
MicroRNA Labelling
The labelling of the microRNA fraction was adapted from a published protocol developed at EMBL (Heidelburg, Germany) by the European Molecular Biology Group (Castoldi et al., “A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA),” RNA 2006 May; 12(5):913-20. Epub 2006 Mar. 15, incorporated herein by reference in its entirety). Briefly, the microRNA fraction was incubated for 6 hours at 4° C. with a mixture containing 10 μM of dye-labelled tetra-nucleotide (5′-rUrUrUrU-Cy5-3′) (or alternatively, 5′-rUrUrUrU-Cy3-3′) (Biospring, Germany) in Ambion buffer diluted to 1× with RNase free water, 8% polyethylene glycol (PEG), 2 mM adenosine triphosphate (ATP), and T4 RNA ligase (0.7 U/μl). The labelling reaction was run by heating the mixture for 15 minutes at 65° C. This procedure ligated the poly-U dye-labelled tail to the 3′ end of all the microRNAs. Labelled samples were stored at 4° C. before hybridization.
Array Hybridization
The labelled microRNA fraction was hybridized to the spotted arrays using a Discovery hybridization station (Ventana, Tucson, Ariz.). Briefly, 2 mL of a mixture of 1% BSA, 2×SSC, and 0.2% SDS was incubated with the chips for 30 min at 42° C. Then the chips were washed once using EZ Prep buffer (Ventana) and then three more times with Ribowash (Ventana). Next, 20 μl of the labelled microRNA mixture and 180 μl of ChipHybe Reagent (Ventana) were added to the array. The arrays were heated for 6 minutes at 37° C., then were incubated at 42° C. for 8 hours, after which the heating was stopped. The chips were washed once with Ribowash (Ventana) and then heated for 2 minutes at 37° C. The chips were washed again with Ribowash (Ventana) with one drop of CheapClean (Ventana) added, and incubated for 2 minutes at 37° C. The chips were washed two more times using Ribowash (Ventana). The chips were stored dry overnight. On the following day, the final washes were done according to Ventana's instructions for the Discovery hybridization station. The slides were washed twice with 2×SSC+0.2×SDS buffer and then one more time with 0.1×SSC. All the slides were dried using a speed centrifuge from Arrayit (TeleChem International, Sunnyvale, Calif.) at room temperature and kept in the dark before scanning.
As an alternative to the ChipHybe Reagent solution (solution 1), the following solution may be used for array hybridization (solution 2) to form probe:target RNA hybrids by mixing 2 parts of the 1.5×TMAC Hybridization Solution to 1 part (v:v) sample, so that the final component concentrations are 3M TMAC, 0.10% Sarkosyl, 50 nM Tris, and 4 mM EDTA, and incubating on the array at 42° C. for 8 h:







TABLE 6







1.5X TMAC Hybridization Solution













Amount/


Reagent
Catalog Number
Final Conc
250 mL





5 M TMAC*
Sigma T3411
4.5 M 
 225 mL


20% Sarkosyl
—
0.15%
1.88 mL


1 M Tris-HCl, pH 8.0
Sigma T3038
75 mM
18.75 mL 


0.5 M EDTA, pH 8.0
Invitrogen
 6 mM
 3.0 mL



15575-020


H2O
—
—
1.37 mL





*TMAC is tetramethyl ammonium chloride






Array Image Acquisition

The arrays were scanned using an AxonTM scanner (Molecular Devices, Sunnyvale, Calif.) and their GenepixTM software. The image was formatted in tif format, defined by an image color depth of 16 bits/pixel (1600*1600). At such setting, pixels can assume intensity values ranging from 0 to 65,535. Pixels exhibiting the maximum intensity value are “saturated” and were assigned the value of 65,535. The resolution of the array scan was set at 10 μn1/pixel. For hybridization experiments using different fluorescent dyes (e.g., Cy5 and Cy3) the photomultiplier tube (PMT) was adjusted to the higher intensity spot (Cy3 is scanned at lower PMT settings than Cy5).
Array Image Analysis
The PMT of the laser scanner digitized the captured fluorescence intensity for each given “point” of a slide and stored the numerical value as a pixel corresponding to that point. A picture composed of such pixels was then analyzed.
The first task for image analysis was to detect the spot position, using a process called segmentation. Spots were segmented by circles of adaptable or fixed radius. To be reliably segmented and quantified, the spot diameter was required to be more than 5-6 pixels. Before segmentation an indexing grid was provided giving the approximate positions of the spots. The segmentation itself detected the limits of spots near the grid circles. Briefly, the Genepix software assigns a circle to each spot on the array (segmentation). The segmentation had to be conducted in a somewhat flexible way due to spotting imperfections and/or support deformation, as the spots were almost never on a perfectly rectangular grid.
After segmentation by the software, the circles were modified manually and adjusted onto the spots until all the spots on the array were clearly identified. At this stage, if the array presented high background noise preventing real spots from being distinguished from the background, the array was rejected for further analysis.
The second task of image analysis was to quantify spots and export the data into a result file. This was a relatively easy and well-defined task once the spots were located on the image. The statistical approach used most frequently to quantify spot intensity was the mean or median of pixels belonging to a spot. The median approach was more robust than the mean value in the presence of outlier pixels. In practice, however, there was little difference in the results obtained using mean or median.
Array Data Analysis
All the array data were analysed using the R bioconductor package (“Bioconductor: open software development for computational biology and bioinformatics,” Genome Biol. 2004; 5(10):R80. Epub 2004 Sep. 15, which is incorporated herein by reference in its entirety).
Array data were first tested for quality by comparing the spot intensities for the internal controls. (Tables 7 and 8) One internal control (SEQ ID NO: 178) was used as a labelling control (this synthetic RNA is added to the purified microRNA fraction before labelling), and 7 other internal controls (SEQ ID NOs: 179-185) were used for the normalization of the data (these synthetic RNA controls are added to the total RNA fraction before hybridization at 520 fmol each/array).







TABLE 7





Internal controls added to total RNA


or microRNA fraction
















CGCGCGUCGCUUUAUCUACUGU
SEQ ID NO: 178; CTL30_COMP


 


UUAUCGUUCGAUAAGUCGCGUU
SEQ ID NO: 179; CTL11_COMP


 


GAAGUUACUAUGUAGGCAACCU
SEQ ID NO: 180; CTL23_COMP


 


CGCGGGACUAAUUGUUACCGGG
SEQ ID NO: 181; CTL26_COMP


 


UCGCGUCGAACUCCGCAACCGA
SEQ ID NO: 182; CTL29_COMP


 


ACCGAACGCCGUACCCAUCGGG
SEQ ID NO: 183; CTL31_COMP


 


CGAGGGUAACGACUCUCGUGUC
SEQ ID NO: 184; CTL36_COMP


 


GCGUACCGACGCGUAGACGGAC
SEQ ID NO: 185; CTL13_COMP
















TABLE 8







Probes for hybridization of control sequences in


microarray experiments









Sequence



identifica-


Sequence (5′-3′)
tion number





TTGTAATACGACTCAACAGTAGATAAAGCGACGCGCG
SEQ ID NO:



186; CTL30


 


TTGTAATACGACTCAAACGCGACTTATCGAACGATAA
SEQ ID NO:



187; CTL11


 


TTGTAATACGACTCAAGGTTGCCTACATAGTAACTTC
SEQ ID NO:



188; CTL23


 


TTGTAATACGACTCACCCGGTAACAATTAGTCCCGCG
SEQ ID NO:



189; CTL26


 


TTGTAATACGACTCATCGGTTGCGGAGTTCGACGCGA
SEQ ID NO:



190; CTL29


 


TTGTAATACGACTCACCCGATGGGTACGGCGTTCGGT
SEQ ID NO:



191; CTL31


 


TTGTAATACGACTCAGACACGAGAGTCGTTACCCTCG
SEQ ID NO:



192; CTL36


 


TTGTAATACGACTCACCCGGTAACAATTAGACCCGCG
SEQ ID NO:



193;



CTL26_MUT


 


TTGTAATACGACTCAGTCCGTCTACGCGTCGGTACGC
SEQ ID NO:



194; CTL13


 


TTGTAATACGACTCAGGCCGTCTACGCGTCGGTACGC
SEQ ID NO:



195;



CTL13_MUT









All sequences for which the intensity of the spot was higher than the mean local background intensity plus 1.5 times its standard deviation were categorized as expressed microRNAs. The following criteria were required to be met in order consider the array intensity data valid for further analysis:

    
    
        1. Specificity of the hybridization controls had to be within acceptance criteria (e.g. CTL26 vs. its corresponding single base mutant, CTL26 MUT, or CTL13 vs. its corresponding single base mutant, CTL13 MUT.
        2. Approximate equality of the signal intensity of the replicates of the positive controls
        3. Approximate equality between median block signal intensities based on the positive controls for each block
        4. Approximate equality between median array signals based on all sequences detected
        5. Signal intensity for the purification and labelling control (CTL30).
    
    


Statistical normalization of the data was done by computing the Log 2ratio where the Log 2ratio equals average intensity signal of the duplicated spots/median intensity of all positives controls for the block. The normalization was done per block to avoid non-homogenous labelling of all blocks of the array. This block-by-block normalization has been shown to be more efficient then using overall normalization of the slide. The obtained values are Log 2 values.
The intensities of the spots for each oligonucleotide probe were compared in the sample from the THP-1 cell line versus normal human monocytes, resulting in an evaluation of the relative expression for each microRNA.
The expression fold-change corresponds to 2(Log 2ratio). The Log 2ratio is the ratio between the two conditions compared, or log 2(Xcell-line/Xnormal), which is the same as (log 2Xcell-line−log 2Xnormal), where X is the measured intensity value. In cases where there was no signal from the “normal” condition, the lowest measured intensity value in the experiment was used as the baseline from which a fold-change expression value was calculated. A fold-change value of less than zero corresponds to a down-regulation of (1/fold-change) times.
Data are tabulated in Table 2, and include all microRNAs over-expressed in monocytes in response to stimulation with one or more TLR agonists.
5.2 Example 2
Analysis of MicroRNAs from Stimulated Monocytes on Luminex Platform
The Luminex technology (Luminex Corp., Austin, Tex.) is based on liquid phase hybridization to probe-labelled beads, followed by flow cytometry detection of beads with differing ratios of fluorescent dyes. Beads with up to 100 different dye ratios are available, making it possible to interrogate a single sample for up to 100 analytes simultaneously.
Monocyte Samples
Human monocytes are isolated form whole blood of healthy donors using CD14 positive magnetic microbead positive selection (according to manufacturer's protocol, Miltenyi Biotec). Isolated monocytes are then cultured with RPMI 1640 medium to a concentration of 2.5 million per milliliter in 6-well plates, in a humidified incubator containing 5% CO2 at 37° C.
Stimulation of Monocytes
To analyze miRNA expression, cells are treated for 8 h or 24 h with the stimuli shown above in Table 5 (Example 1).
Isolation of Total RNA from Monocyte Samples
Cells from two confluent 75 cm2 flasks are harvested (=approx 107 cells) for each agonist used to stimulate the monocytes. Total RNA is prepared using TRIzol® Reagent, Invitrogen (Carlsbad, Calif.) according to the manufacturer's protocol. All RNA samples are diluted in RNase-free water and stored in −80° C. (−112° F.).
Coupling of Probes to Luminex Beads
Aliquots of each 5′-amino-modified probe (having the structure 5′AminoC6-probe sequence, i.e., similar to the structure in Example 1, but without the linker sequence) are prepared at a concentration of 0.1 mmol/μL in molecular biology grade water. The probes are coupled to the beads using carbodiimide chemistry according to the manufacturer's protocol (Luminex bead coupling protocol). The probe-coupled beads are stored at 4° C.
Total RNA Preparation for Luminex Analysis
Eight fmoles of each of 7 internal controls (the same synthetic RNAs used for the array controls) are added to the total RNA fraction isolated from the patient samples. For each sample, three replicates are assayed in parallel. For each replicate, 250 ng of total RNA is used. Prior to hybridization with Luminex beads, the total RNA preparation is treated with calf intestinal phosphatase (CIP; Invitrogen) to prevent the formation of dendrimers, which result from the circularization of a single RNA molecule, or concatenation to another RNA molecule. Pre-treatment with CIP is according to the manufacturer's protocol, and removes 5′-phosphate groups.
Bead Labelling and Hybridization
After CIP treatment, the total RNA fraction is labelled with biotin using the Vantage microRNA Labelling Kit (Marligen). The labelled fraction is hybridized to the Luminex beads using the Marligen protocol. Briefly, the polynucleotide beads are mixed with the Marligen hybridization solution (1.5×TMAC) and the labelled total RNA. The hybridization is performed at 60° C. for an hour in the dark. After hybridization, the beads are washed using the Luminex standard 6×SSPET wash buffer (sodium phosphate, sodium chloride, EDTA, Triton X-100, pH 7.4).
Detection of Bead Hybridization
The detection of the Luminex beads is done using streptavidin phycoerythrin (SAPE) (Europa Bioproducts, Cambridge, UK). The SAPE is added to the washed beads according to the Luminex protocol. The beads are then read using the Luminex IS-200 instrument on the high gain setting for better resolution.
Data Acquisition and Analysis
The Luminex IS-200 reads at least 25 beads of each dye-ratio in the reaction mix. Each dye-ratio bead corresponds to a particular probe sequence, and the intensity value is returned as an average value of all read beads. The mean fluorescence intensity (MFI) data is normalized using synthetic RNA controls or alternatively using the mean of expressed oligonucleotides, and fold changes between normal and stimulated or diseased samples are computed using the Bioplex software (Bio-Rad, Hercules, Calif.) and the R bioconductor package (Bioconductor: open software development for computational biology and bioinformatics, Genome Biol. 2004; 5(10):R80. Epub 2004 Sep. 15).
Table 9 lists exemplary internal controls RNAs that can be added to total RNA prior to hybridization to the beads. Table 10 shows the corresponding probe sequences that are coupled to control beads.







TABLE 9





Internal controls added to total RNA fraction
















CGCGCGUCGCUUUAUCUACUGU
SEQ ID NO: 178; CTL30_COMP


 


UUAUCGUUCGAUAAGUCGCGUU
SEQ ID NO: 179; CTL11_COMP


 


GAAGUUACUAUGUAGGCAACCU
SEQ ID NO: 180; CTL23_COMP


 


CGCGGGACUAAUUGUUACCGGG
SEQ ID NO: 181; CTL26_COMP


 


UCGCGUCGAACUCCGCAACCGA
SEQ ID NO: 182; CTL29_COMP


 


ACCGAACGCCGUACCCAUCGGG
SEQ ID NO: 183; CTL31_COMP


 


CGAGGGUAACGACUCUCGUGUC
SEQ ID NO: 184; CTL36_COMP


 


GCGUACCGACGCGUAGACGGAC
SEQ ID NO: 185; CTL13_COMP


 


CGCGAUAAACGCCGGAUGGACC
SEQ ID NO: 944; CTL28_COMP


 


UCGAGCGACUCCCGUAAUUUAA
SEQ ID NO: 945; CTL35_COMP
















TABLE 10







Probes for hybridization of control sequences in


Luminex experiments









Sequence


Sequence (5′-3′)
identification number





ACAGTAGATAAAGCGACGCGCG
SEQ ID NO: 934; CTL30b


 


AACGCGACTTATCGAACGATAA
SEQ ID NO: 935; CTL11b


 


AGGTTGCCTACATAGTAACTTC
SEQ ID NO: 936; CTL23b


 


CCCGGTAACAATTAGTCCCGCG
SEQ ID NO: 937; CTL26b


 


TCGGTTGCGGAGTTCGACGCGA
SEQ ID NO: 938; CTL29b


 


CCCGATGGGTACGGCGTTCGGT
SEQ ID NO: 939; CTL31b


 


GACACGAGAGTCGTTACCCTCG
SEQ ID NO: 940; CTL36b


 


CCCGGTAACAATTAGACCCGCG
SEQ ID NO: 941; CTL26b_MUT


 


GTCCGTCTACGCGTCGGTACGC
SEQ ID NO: 942; CTL13b


 


GGCCGTCTACGCGTCGGTACGC
SEQ ID NO: 943; CTL13b_MUT


 


GGTCCATCCGGCGTTTATCGCG
SEQ ID NO: 946; CTL28


 


TTAAATTACGGGAGTCGCTCGA
SEQ ID NO: 947; CTL35









Analysis

All sequences for which the intensity of the coupled bead is higher than 50 MFI are categorized as expressed microRNAs. The following criteria must be met in order consider the coupled bead intensity data valid for further analysis:

    
    
        1. Approximate equality of the signal intensity of the replicates of the positive controls;
        2. Approximate equality between median well signal intensities based on the positive controls for each well;
        3. Approximate equality between median wells signals based on all sequences detected.
    
    


Statistical normalization of the data is done by computing the Log 2ratio where the Log 2ratio equals average intensity signal of 50 replicates of the same bead (each coupled to the same oligo sequence) divided by the median intensity of all of the positive controls in one well. The normalization is done per well to avoid non-homogenous labelling of all wells of the plate. This well-by-well normalization has been shown to be more efficient then using overall normalization of the plate. The obtained values are Log 2 values.
The intensities of the beads for each coupled bead are compared in the sample from monocytes from healthy donors grown in PMI medium, which does not activate the cells, versus monocytes stimulated in vitro (stimulated with a TLR agonist or grown in PMA medium, which activates the cells), resulting in an evaluation of the relative expression for each microRNA.
The expression fold-change corresponds to 2(Log 2ratio). The Log 2ratio is the ratio between the two conditions compared, or log 2(Xcell-line/Xnormal), which is the same as (log 2Xcell-line−log 2Xnormal), where X is the measured intensity value. In cases where there is no signal from the “normal” condition, the lowest measured intensity value in the experiment is used as the baseline from which a fold-change expression value was calculated. A fold-change value of less than zero corresponds to a down-regulation of (1/fold-change) times. A two-fold change, either upregulated or downregulated, is considered significant.
5.3 Example 3
Bioinformatic Analysis to Identify microRNAs
In order to identify the microRNAs detected with the probes shown, e.g., in Table 2, small RNA sequencing (smRNASeq) datasets were analysed using the probe sequences to identify expressed microRNAs detected by those sequences. The analysis identified 11 sequences with precise ends, corresponding to 11 arms. Those 11 candidate microRNA sequences are show in Table 11.







TABLE 11







microRNA candidate sequences


corresponding to probes









Arm name
microRNA candidate sequence 5′ -> 3′
SEQ ID





4214-R
CCCCTGCAGAGCTCACA
215


 


6511-R
AATAGATATTATGTTTTA
216


 


7997-L
TAGTGTAACGGAAATGTTTACA
217


 


6433-L
AAGGCTGAGCAGCAGAGGCAGCAAGAGC
218


 


3995-L
TGGCCTGACGTGAGGAGG
219


 


6192-L
TGGGTGGGTGGTTTTTT
220


 


6998-L
TTCTCCACTGTGCTGCTACC
221


 


9654-L
AGCTATGCTCACTCTCAA
222


 


4504-L
AAATCAATAAATAATCAG
223


 


5230-L
TGTGTTGGGTGACCCTGG
224


 


4440-L
GCCCAGTGCTCTGAATGTCAAA
225









5.4 Example 5
Sequencing Analysis to Identify microRNAs Associated with Sepsis
Total RNA from a sepsis patient was used for preparing a smRNASeq dataset. Briefly, 5 μg of total RNA was used for small RNA sequencing on a Solexa GA 11 (Illumina) using a standard library and sequencing protocol provided by the manufacturer.
The number of times a microRNA appeared in the sepsis smRNASeq dataset was then compared to the number of times the same microRNA appeared in each of 77 non-sepsis patient smRNASeq datasets. When the sepsis smRNASeq dataset contained the highest number of counts for a particular microRNA, it was assigned a rank of 1. When the sepsis smRNASeq dataset contained the second highest number of counts for a particular microRNA, it was assigned a rank of 2, and so on. All candidates having a rank between 1 and 5 were retained. A total of 175 candidate microRNA sequences, corresponding to 165 different aims, are shown in Table 12, along with the rank assigned to each sequence. When a microRNA has multiple isomirs, the sum of all of the counts for all of the isomirs was used for the comparison. Furthermore, when a precursor gene for a particular microRNA sequence is present at multiple locations in the genome, both candidate names are shown, with the same ranking and same sequence. Either or both of those candidates may be present at increased levels in the sepsis patient sample.







TABLE 12







Novel microRNAs found at higher numbers in


a sepsis smRNASeq dataset










microRNA





Candidate
MicroRNA Candidate

SEQ ID


Name
Sequence 5′ -> 3′
Rank
NO





11253-L
CTGTAATTAGTCAGTTTTCTGT
1
226


 


11312-L
GTGGACTCCAGCAGTAG
1
227


 


13196-R
AGAGCTCTCTGGCTTTGCCTTAAA
1
228


 


13339-L
ACCCTCAGTCCGTATTGGTCTCT
1
229


 


13367-L
TGCTGTATTGTCAGGTAGTGA
1
230


 


13446-R
AGCTTTTGGGAATTCAGGTAG
1
231


 


13448-L
TATGTATGTATGTATGT
1
232


 


13452-L
TATGGAGGTTCTAGACCATGT
1
233


 


13627-L
ATCCTAGCTTGCCTGAGACTGT
1
234


 


13629-L
TCTGATCAGGCAAAATTGCAGA
1
235


 


13642-R
AAAAACTGTGATTACTTTTGCA
1
236


 


13661-R
GAGACAGTAGTTCTTGCCTGGT
1
237


 


13665-R
TCTCTTTATATGTACTGGAGC
1
238


 


13667-R
TAAAAACCGTGACTACTTCTG
1
239


 


13670-L
ATAGGACTTTTGAAGGAAGAG
1
240


 


13672-R
AAATGTTGAGATACACTGAA
1
241


 


13677-L
AATTACAGATTGTCTCAGAGAA
1
242


 


13691-R
AAAATCCTTTTTGTTTTTCCAG
1
243


 


13692-R
ATTTAACTGGACATCTTGCATT
1
244


 


13694-L
ACCTGGACCCAGCGTAGACAAAGA
1
245


 


13694-R
CATATCTACCTGGACCCAGTG
1
246


 


13694-R
CATATCTACCTGGACCCAGTGTA
1
247


 


13696-L
ATCCTAGCTTGCCTGAGACTGT
1
248


 


13716-L
AAGAGTTACTAGAACTATT
1
249


 


13716-R
GAATAGTTTTAGTAACTCTTGA
1
250


 


13717-L
AATTTATTCTTGGTAGGTTGTA
1
251


 


13717-R
CAACGTACTAAGAATAAATTTC
1
252


 


13719-L
AGCTCTAGAAAGATTGTTGACCA
1
253


 


13721-R
ATTGTTTAGTACCTATAATG
1
254


 


13727-L
CGGAGTTGTAAGTGTTGACAA
1
255


 


13727-R
ACTTACAACTCTGGAAAAGCAGA
1
256


 


13729-L
AGCAATACTGTTACCTGAAATA
1
257


 


13731-L
GAAGAACTGTTGCATTTGCCCTG
1
258


 


13755-L
TAGTGGTCAGAGGGCTTATGA
1
259


 


14086-L
GGTGTAATGGTTAGCACTCTGGA
1
260


 


14093-L
AGTGGTAGAGCATTTGA
1
261


 


14111-L
GTAGTGTTTCTTACTTTA
1
262


 


14113-L
AGATCGCCGAAGCGTCGGA
1
263


 


14117-R
TTAAAACTTTAAGTGTGCCTA
1
264


 


14154-L
AGTTTCTAAGGATCATGTCTG
1
265


 


14177-L
TAGTGGTTAGTACTCTG
1
266


 


14226-R
AGTTTCTAAGGATCATGTCTG
1
267


 


14229-R
ACCTTCACTGTGACTCTGCTG
1
268


 


14371-R
GTTTCCGTAGCGTAGTGGTTATCA
1
269


 


14375-R
AGTCCTTAACAAGCATTGAGA
1
270


 


14390-L
GTTAAGTGCTCCAAGGAGGTGG
1
271


 


14390-L
GTTGCTATCGGGGACTAC
1
272


 


14399-L
GCTGTGCTACGTCGCCCTGGA
1
273


 


14479-R
ATTTTTCTTATAGGCTTCTAAG
1
274


 


14482-R
GAATATGGGTATATTAGTTTGG
1
275


 


14499-L
GGCCAGCCACCAGGAGGGCTGC
1
276


 


14570-R
ACTAATAGAGGTAATAGTTGAA
1
277


 


14596-R
TAGGAGGGAATAGTAAAAGCAG
1
278


 


  182-R
GACGTCAGAGGGAATCC
1
279


 


 2851-R
CAGAAGGGGAGTTGGGAGCAGA
1
280


 


 4215-L
TATGAACAGTGGATAGATTAAAGG
1
281


 


 4417-L
ATCGGAGAAACTCCCTGCGATGAG
1
282


 


 4784-L
CATCTTTTATTTGGTAAATTATGA
1
283


 


 4784-R
ATTGATTGTGGCAAAGTT
1
284


 


 4784-R
TATATTGATTGTGGCAAAGTT
1
285


 


 5392-R
ATGAGATACTGTCGGAGA
1
286


 


 6415-R
ATTGTCCTTGCTGTTTGGAGATA
1
287


 


 7038-R
ATTTTCTGAACTGTACAT
1
288


 


 7491-R
GTGGATTTTGTTTGCTGT
1
289


 


11626-R
ACTGGCCTGGGACTACCGGGGG
2
290


 


13270-L
TTGTTCTTTGGTCTTTCAGC
2
291


 


13322-R
CATTACTGATTTTCTTTTCTTAGA
2
292


 


13375-L
AGGGGCTGGGGTTTCAGGTTCT
2
293


 


13375-L
CAGGGGCTGGGGTTTCAGGTT
2
294


 


13400-R
TTGTCTCTTGTTCCTCACACAG
2
295


 


13437-R
ATATTATTAGCCACTTCTGGAT
2
296


 


13446-L
ACCTGAATTACCAAAAGCTTT
2
297


 


13447-R
TATGGAAAGACTTTGCCACTCT
2
298


 


13448-R
TATGGAAAGACTTTGCCACTCT
2
299


 


13465-R
TAACGCATAATATGGACATGT
2
300


 


13531-R
AGACTGACCTTCAACCCCACAG
2
301


 


13629-R
CCTGCAACTTTGCCTGATCAGA
2
302


 


13640-L
TGGATATGATGACTGAA
2
303


 


13686-L
ACTTGTAATGGAGAACACTAAGC
2
304


 


13687-R
ATTAAGGACATTTGTGATTGAT
2
305


 


13704-R
TTTTTGTCAGTACATGTTAATG
2
306


 


13710-R
ATTTGCTGTTAAGATATGGGAT
2
307


 


14113-R
ACTACCGTTGGTTTCCGC
2
308


 


14151-R
AGAGTTTGGATTAGTGGG
2
309


 


14170-L
AACTAGCTCTGTGGATCCTGAC
2
310


 


14220-L
TACGGATAATTGTAGCACTTC
2
311


 


14327-R
AATGTTGGAATCCTCGCTAGAGC
2
312


 


14328-R
TGTGGGACTTCTGGCCTTGACT
2
313


 


14347-R
ACCTGGGTTGTCCCCTCTAG
2
314


 


14361-L
GCCTGGACTGCCTGGAGAAAGCG
2
315


 


14385-L
GTGGCCGAGGACTTTGA
2
316


 


14385-L
GTGGCCGAGGACTTTGATT
2
317


 


14385-L
GTGGCCGAGGACTTTGATTG
2
318


 


14415-R
TTAAGATTTGGTGCAATAT
2
319


 


14483-R
ACTCTTTAAGGATAGGGCTGAA
2
320


 


14508-L
TAGGGGAAAAGTCCTGATCCGG
2
321


 


14527-R
TAGACAATCTGTGTAGAGTGC
2
322


 


14532-L
TGAGACCAGGACTGGATGCACCA
2
323


 


14539-R
AAGTTTCTCTGAAGGTGTAG
2
324


 


14539-R
AGTTTCTCTGAAGGTGTAG
2
325


 


14541-R
ATTAAGGACATTTGTGATT
2
326


 


14549-R
TTGTAACATTCTGGTGTGTTG
2
327


 


14578-R
TTGTAACATTCTGGTGTGTTG
2
328


 


   62-L
GAATTAATGGCTGGCTGGGAG
2
329


 


 6520-R
ATATTGGAATCCCCGCTAGAGC
2
330


 


 7578-L
GGGGCTGTAGCTCAGGG
2
331


 


11607-R
GGAGGAACCTTGGAGCTTCGGC
3
332


 


12625-R
GCAGCCCAGCTGAGGCCTCTG
3
333


 


13005-R
AACAACTTAGACACGTGACTGTA
3
334


 


13214-L
TTCCTTAACTAAAGTACTCAGA
3
335


 


13244-R
CAGGCAGCTGTTAACAG
3
336


 


13340-L
GAAGCAGCGCCTGTCGCAACTCG
3
337


 


13452-R
TACATGGATGGAAACCTTCAAGCA
3
338


 


13628-L
AGACCCATTGAGGAGAAGGTTC
3
339


 


13655-L
CAAAAGTGATCGTGGTTTTTG
3
340


 


13677-R
TCTCTCGGACAAGCTGTAGGT
3
341


 


14085-L
CTAAGCCAGGGATTGTGGGT
3
342


 


14156-R
CTTACACTCTTGTCCATCTAGA
3
343


 


14161-R
TGAACAGCCTCTGGCAATC
3
344


 


14244-R
TCACAATGCTGACACTCAAACTGCTGACA
3
345


 


14289-R
ATGTAGTCTCCCCTACCTAG
3
346


 


14380-R
CATGAGATCCAACTCTGAGC
3
347


 


14409-R
TGAGTTTAGAGCTGTCTGCT
3
348


 


14469-R
GACACCTCTGCACTCAAGGCGG
3
349


 


14469-R
GACACCTCTGCACTCAAGGCGGC
3
350


 


14485-L
GCAAGAAAGTGAGACTCTGCCT
3
351


 


14556-R
AAGTTTCTCTGAACGTGTA
3
352


 


14567-R
CATGCTAGGATAGAAAGAATGGG
3
353


 


14581-L
TATCCAGCTTGTTACTATATGC
3
354


 


 2970-L
CCTCCCAAAGTGCTGGGATTA
3
355


 


  607-R
AATGATGATGCAAGAACTGAGA
3
356


 


 6493-L
TAGGAGCTATCAGAACTTAGTG
3
357


 


11549-L
GTTCCTGCTGAACTGAGCCAGT
4
358


 


11667-L
GTTCCTGCTGAACTGAGCCAGT
4
359


 


12627-R
CCAACTAACCTCTGTATTCCAG
4
360


 


13374-L
TGTTCCTCTGTCTCCCAGACTCTG
4
361


 


13681-R
TGAGGAGATCGTCGAGGTTGGC
4
362


 


13705-L
CCTCCCACTGCAGAGCCTGGG
4
363


 


14069-R
GCCGGGTACTTTCGTATT
4
364


 


14137-R
TTGGCTGGTCTCTGCTCCGCAG
4
365


 


14200-L
CCTCCCAAAGTGCTGGGATTACAGGC
4
366


 


14244-L
GTTGGAGGATGAAAGTA
4
367


 


14275-L
TGCCTTAGGAGAAAGTTTCTGG
4
368


 


14288-R
GCCCGGAGAGCTGGGAGCCAGA
4
369


 


14303-R
ACCGGATGGAGCTCTAGGGA
4
370


 


14332-L
TGCCTTAGGAGAAAGTTTCTGG
4
371


 


14367-R
ATGGATGATGATATCTGAGT
4
372


 


14452-L
TTCCTTAACTAAAGTACTC
4
373


 


14543-L
GAGCCAGTGGTGAGACAGTGAG
4
374


 


14601-L
CGGGGCTGCCCTGAACGGGCCC
4
375


 


 2999-R
GTTCGAGACCAGCCTGGCC
4
376


 


  373-R
GTCGAACTTGACTATCTAG
4
377


 


 6216-L
GTGCTCTGAATGTCAAAGTGAAGA
4
378


 


13413-R
CAGTTCAATGGTGTTCAGCAGA
5
379


 


13630-L
AAAAGTAGTTGTGGTTTT
5
380


 


13679-L
AAAAGTAGTTGTGGTTTT
5
381


 


13709-R
AAGGGCTTCCTCTCTGCAGGAC
5
382


 


13734-L
TGGACTTTTTCAGATTTGGGGAT
5
383


 


13745-L
GACTCACTCACAGGATTGTGCA
5
384


 


13748-L
GACTCACTCACAGGATTGTGCA
5
385


 


13753-L
CCACTTGGATCTGAAGGCTGCC
5
386


 


13761-L
TGAGGAGGATCTGAAGGATTGGA
5
387


 


14121-R
TCATGTTGCTCTGCTGTTGCC
5
388


 


14336-R
AGGAGAATCACTTGAACCC
5
389


 


14338-R
ATGAAGTGTGACGTGGAC
5
390


 


14338-R
TGGACATCCGCAAAGACCTGTACGCC
5
391


 


14442-R
GCCTTTTTAACCGCGAGCGACA
5
392


 


14454-R
ATGAAGTGTGACGTGGAC
5
393


 


14454-R
TGGACATCCGCAAAGACCTGTACGCCAA
5
394


 


14494-L
AGGACTGGACTCCCGGCAGCC
5
395


 


14575-R
AAGGGCTTCCTCTCTGCAGGAC
5
396


 


14583-L
ATGGCCAGAGCTCACACAGAGG
5
950


 


14584-L
ATGGCCAGAGCTCACACAGAGG
5
397


 


 2850-L
AAAGTGCTGGGATTACAGG
5
398


 


4135-R
GATGCCTGGGAGTTGCGATCTG
5
399









Predicted microRNA precursor sequences for the microRNAs listed in Table 12 are shown in Table 13.







TABLE 13







Precursor sequences and chromosomal locations (from human genome NCBI36)


of the microRNAs in Table 17



















SEQ


microRNA





ID


Candidate
microRNA precursor Sequence 5′->3′
chrom
start
end
strand
NO
















   62-L
GCAAAAGACATTTCCTTAGAATTAATGGCTGGCTGGGAGGCAGAGCCAAGGGC
12
64538072
64538169
−1
400



CACTGGTTCCTCCCAGCTGGTCATTAATCCTCAGGAAATGCCTGC







 


  182-R
GGAGTTCCTGTGACGCAATTAGCCATATAAGGAGCTCGGCCGGCGCGGCGGAG
19
50662759
50662851
−1
401



TGTTTGTTTGGTATCCTAGCAATGACGTCAGAGGGAATCC







 


  373-R
TTGGATGGTTTAGTGAGGCCCTCGGATCAGCCCGCTGGGTCAGCCCACTGCCC
2
132728445
132728537
−1
402



TGGCGGAACGCTGAGAAGACAGTCGAACTTGACTATCTAG







 


  607-R
GGGGGCTCATTAGGTGGAGATTGGTTTTTAATCAGCACCATGGCAATGATGAT
1
158601772
158601859
1
403



GCAAGAACTGAGATGCCACCTGATCCCCTGCCCCC







 


 2850-L
CAAAGTGCTGGGATTACAGGCGTGAGCCCCCGTGCCCGGCCTGTGTGTTTTTT
19
10794434
10794541
−1
404



ATAAAAAAAGTTTTGGCTGGGCACAGTGGCTCACGCCTGTAATCCCAACACTT








TG







 


 2851-R
CCCTTCCTGCCGGCCGGCCCCCTCCTCAGGCCCCTCCTTCTCAGCCCCAGCTC
9
130047027
130047158
−1
405



CCGCTCACCCCTGCCACGTCAAAGGAGGCAGAAGGGGAGTTGGGAGCAGAGAG








GGGACCACGGGCTGGCTGGTCTGGGG







 


 2970-L
GCCTCCCAAAGTGCTGGGATTATAGGCGTGAGCCACTACGCCCAGCCTCTGAA
19
58991703
58991827
1
406



ACATTTTAAAAAATTATCTGGGTTAGGCCGGGCGTGGTGGCTCACGCCTGTAA








TTCCAGCACTTTGGGAGGC







 


 2999-R
GCCCAGGCTGCTGTCAAACTCCTGAGCTCACCCAGGCGCAATGGCTCATGCTT
11
74716486
74716607
−1
407



GTAATCCCAGCACTGGGAGGCCAAGGTGGGCAGATCACCTGAGGTCAGGAGTT








CGAGACCAGCCTGGCC







 


 4135-R
CGGGTAAAGGTCGCCCTCAAGGTGACCCGCCTACTTTGCGGGATGCCTGGGAG
22
41341277
41341343
1
408



TTGCGATCTGCCCG







 


 4215-L
GGCTTACAGAATTATGAACAGTGGATAGATTAAAGGCATTTAATATTTGTAAT
12
104086228
104086303
1
409



TCATAATAACTGTAGAAATGGCC







 


 4417-L
GCTGGGGTTCATCGGAGAAACTCCCTGCGATGAGCCACTAGGGTCACGGACAG
14
34943859
34943935
1
410



GGAACTTTTTGATGAGCGCCGAGT







 


 4784-R
AACTTTGTTAATCATCTTTTATTTGGTAAATTATGAATGGGTATACATTTGTA
12
9180205
9180287
1
411



CAGTTCGTGTATATTGATTGTGGCAAAGTT







 


 4784-L
AACTTTGTTAATCATCTTTTATTTGGTAAATTATGAATGGGTATACATTTGTA
12
9180205
9180287
1
412



CAGTTCGTGTATATTGATTGTGGCAAAGTT







 


 4784-R
AACTTTGTTAATCATCTTTTATTTGGTAAATTATGAATGGGTATACATTTGTA
12
9180205
9180287
1
413



CAGTTCGTGTATATTGATTGTGGCAAAGTT







 


 5392-R
CTCTCTCTCTCAGTTACTCACAAAACATGGCTGTCTTATTCAGAGATTAGCAA
2
160081288
160081369
1
414



TTATTGTAATGAGATACTGTCGGAGAGGG







 


 6216-L
CATGTGATTTCTGCCCACTGCTCTGAATGTCAAAGTGAAGAAATTCAATGAAG
11
77275152
77275231
1
415



CACGGGTAAACGGCGGGAGTAACTATG







 


 6415-R
AGAAAATAACATTGTCAGACGTGTCATCCCCAGATACAATGGACAATATGCTA
2
207682945
207683057
−1
416



TTATAATCGTATGGCATTGTCCTTGCTGTTTGGAGATAATAGTGCTGACTTTA








TTCCTCT







 


 6493-L
TTATTTACCAGCTCAGAATGTGGTAGGAGCTATCAGAACTTAGTGATCAAGTG
1
169826822
169826930
1
417



AAGTCGTAGTTACTAATTTCTGATGCTCTTCCCCTGCAGAAGAGAGCTGTGGG








AAG







 


 6520-R
GCTTCCAGAAGTGCACGCTCTAGCGAGGATTCCAACATTGGGGGAATTGGCCC
1
54292336
54292423
−1
418



AATATTGGAATCCCCGCTAGAGCGTGCACCTAAGC







 


 7038-R
ATTTAAAGAGAATTTTATTTACAGTTATTTCAAATGTAGAGAATGTAATTTTC
18
28363735
28363816
1
419



TGAACTGTACATAAGTGCTCAATTTAAAT







 


 7491-R
TCATTAATTTATGCAAGGTAGCAGCCAGCATATTAGTTCACACCATTTGTGGA
5
115808968
115809054
1
420



TTTTGTTTGCTGTCTCACATGCAAGGTGAAATGG







 


 7578-L
GAGGGGCTGTAGCTCAGGGTGTGCACTGCGAGGCTGGACCTGTTGAGTCTGCA
2
104755260
104755340
1
421



GTGGACATCCATTTAGCTTCAGGTTGTC







 


11253-L
GGCGTGCCTGGGAGTTGGCACTAAGTACAGCTGTAATTAGTCAGTTTTCTGTC
1
234082900
234083009
−1
422



CTGTCCACACAGAAAACCGTCTAGTTACAGTTGTAAGTTGTGCCAGACCTAAT








CGCT







 


11312-L
CCACCAACCTGTGGGAAGGCAGGTGGACTCCAGCAGTAGGTAGGTAACATCCA
6
90036688
90036793
−1
423



AGGTAGCCATACACAGCTGGTGTTTGTGAATCTGCACTTCCCCTGGGCAATGG







 


11549-L
GGGCTTGACCCCTGTTCCTGCTGAACTGAGCCAGTGTACACAAACCAACTGTG
19
13808092
13808176
1
424



TTTCAGCTCAGTAGGCACGGGAGGCAGAGCCC







 


11607-R
GCTGAAGCTCTAAGGTTCCGCCTGCGGGCAGGAAGCGGAGGAACCTTGGAGCT
22
29886048
29886105
−1
425



TCGGC







 


11626-R
ATGGCCTCTCCAGTCTGCAGCTCCCGGCAGCCTCGGGCCACACTCCCGGGATC
16
533277
533300
1
426



CCCAGGGACTGGCCTGGGACTACCGGGGGTGGCGGCCGTGGCTCTGGCTAT







 


11667-L
GGCTTTTTGCTCAAGGGCTCGACTCCTGTTCCTGCTGAACTGAGCCAGTGTGT
9
96888104
96888213
−1
427



AAAATGAGAACTGATATCAGCTCAGTAGGCACCGGAGGGCGGGTCCAATCGAC








AGCC







 


12625-R
CCACACCTGAGAACACGGCAGGTGAGCAGGACGAGGCTGGGCTGAACCCGTGG
11
65160334
65160444
1
428



GTGAGGAGTGCAGCCCAGCTGAGGCCTCTGCTGTCTTATCTGTCTCCTACAGG








CAATGG







 


12627-R
CCACCTGGTTCCTGTACCCGTTCAGACGGTTCTTTGCAAGACATTCCTCCCAT
17
75962769
75962856
1
429



TGTTCACCCCCAACTAACCTCTGTATTCCAGGGGC







 


13005-R
TACAGTTCTGTCTAGGCAGTGGCTTGGGTTTTTATCGAGCAACAACTTAGACA
12
66342382
66342539
1
430



CGTGACTGTAATATGCTGCAACTGTGTGTACTGAAAATATGTGAAAATGGTTG








AATGTGGACTGTGTATATATGTATGTAAAAATTTCTGTGAGATGCTGCTGTC







 


13196-R
AGGTGCTCCACCGTTCCTGCTGTGGAGAAGGAGGCGAAGTCAGAGAGCTCTTC
10
103976793
103976909
1
431



CAAGCTTTCCCCAGGAAGAGCTCTCTGGCTTTGCCTTAAAGCTCCCCAGAGGT








TTTGGAGGCTG







 


13214-L
ATTTCTTTCCTTAACTAAAGTACTCAGATATTTATCCAAACATTATTGCTATG
10
89643735
89643853
1
432



GGATTTCCTGCAGAAAGACTTGAAGGCGTATACAGGACAATATTGATGATGTA








GTAAGGTAAGAA







 


13244-R
GCTGTGCTGTGTGCCAATGTTTCGTTTGCCTCAGACAGGTATCTCTTCGTTAT
11
65027130
65027238
1
433



CAGAAGAGTTGCTTCATTTCATCTGGGAGCAGAAAACAGCAGGCAGCTGTTAA








CAG







 


13270-L
TGGATGATTTGGAGTAGCAAAGCAGCAATTGTTCTTTGGTCTTTCAGCCATGA
1
241576085
241576188
1
949



CCTGACCTTCTGTCTGTGAGACCAAAGAACTACTTTGCTTGGCCACCATCT







 


13322-R
TTCTAGAACCGTAAAAAAGGAAGTAAGTACTGGTACATTACATTTTACACTTC
16
51818856
51818973
1
434



ATATTCTGTGGCCTGTTTAAAAGAGTCACCATTACTGATTTTCTTTTCTTAGA








TATCACCTGGAG







 


13339-L
GCGGCGGACACCATCTTCTTTAAACCCTCAGTCCGTATTGGTCTCTATGGCAT
17
7150842
7150954
−1
435



CCATAGAGGCCATTCGGCTCTGAGGTCCTCAGTAAAGAAACTTAGATGGTATT








ACTGTGT







 


13340-L
CTCGTGGGAAGCAGCGCCTGTCGCAACTCGCCACTTGTTCTCCTCACAGCAGG
17
73648461
73648551
1
436



TTCAGGAGAAGTGGCACCTGGTGGAGGACCTGTCGCGA







 


13367-L
TATAAAAATAGGTAATATAGAAAATAAAACACATACTGCTGTATTGTCAGGTA
19
8360164
8360280
−1
437



GTGATAGGATTTATCACTACCTGACAATACAGTATGTGTTTGTTTTATATATT








TAGGGTGTACA







 


13374-L
TGGCGCTGCTCTGCTGTTCCTCTGTCTCCCAGACTCTGGGTGGATGGAGCAGG
20
61388601
61388684
1
438



TCGGGGGCCAGGGGACAGGAAGGCTAGGGCC







 


13375-L
ACCCTCTCAGGACCCCTCCTAAGGGGTAGGCAGGGGCTGGGGTTTCAGGTTCT
20
62178716
62178832
−1
439



CAGTCAGAACCTTGGCCCCTCTCCCCAGACCCCCAGGCTGTGGTGAGGGTCTG








AGAGCTGGTAC







 


13400-R
ATCTATTTTGTGTGAGTACAGAGAGCATCTGAATGGGTACAGTTGTTGTCTCT
22
28733036
28733110
1
440



TGTTCCTCACACAGGCACCAGA







 


13413-R
GGATTGACATCGCTAGATTTCCCATTACTGTTGCTGAATCCAGAGAGCAGCAT
2
74619921
74620039
−1
441



GAGTTCCTGAAATGCAGTTCAATGGTGTTCAGCAGACGGTGGATGCGGAAAAT








CTTCATTTTTCCC







 


13437-R
ATCTCTGATATGGAAGAAATCCAGAAGTGGCTAATAATATTGACACTATAACA
3
87358016
87358122
−1
442



ATAATGTCAATATTATTAGCCACTTCTGGATTTATGAATCATGTCTCAAGG








ATT







 


13446-L
TGGAAGCCTACCATTTATGTCCTCTTGAGGTACCTGAATTACCAAAAGCTTTAT
4
153629918
153630038
−1
443



GTATTCTGAAGTTATTGAAAATAAGAGCTTTTGGGAATTCAGGTAGTTCAGGAG








TGACTTTTCTAAA







 


13446-R
TGGAAGCCTACCATTTATGTCCTCTTGAGGTACCTGAATTACCAAAAGCTTTAT
4
153629918
153630038
−1
444



GTATTCTGAAGTTATTGAAAATAAGAGCTTTTGGGAATTCAGGTAGTTCAGGAG








TGACTTTTCTAAA







 


13447-R
CCAAAGTTCTTCACTTTAAAGAGTGGCAAAGTCTTTCCATATGTGTAACAGACA
4
160269396
160269504
1
445



TACATACATACATATGGAAAGACTTTGCCACTCTTGAAAGTGAAGAGTGTGTGT







 


13448-R
TCTTCACTTTCAAGAGTGGCAAAGTCTTTCCATATGTATGTATGTATGTCTGTT
4
160269402
160269496
−1
446



ACACATATGGAAAGACTTTGCCACTCTTTAAAGTGAAGAAC







 


13448-L
TCTTCACTTTCAAGAGTGGCAAAGTCTTTCCATATGTATGTATGTATGTCTGTT
4
160269402
160269496
−1
447



ACACATATGGAAAGACTTTGCCACTCTTTAAAGTGAAGAAC







 


13452-R
TTCATTCTATTGAGCTGACTGGCTTGTATGGAGGTTCTAGACCATGTTAGTGTT
4
40198797
40198907
−1
448



CAAGTCTACATGGATGGAAACCTTCAAGCAGGCCAAGCAGGAGACAGGTGGAAG








AAG







 


13452-L
TTCATTCTATTGAGCTGACTGGCTTGTATGGAGGTTCTAGACCATGTTAGTGTT
4
40198797
40198907
−1
449



CAACTCTACATGGATGGAAACCTTCAAGCAGGCCAAGCAGGAGACAGGTGGAAG








AAG







 


13465-R
GTCTATCAAGAGAGGAATGAACAGTTAAATTATAACATGTCCATATTATGGGTT
5
170746260
170746377
−1
450



AGTTGTGGACACATACTAACGCATAATATGGACATGTTATAATTTAACTGTTCC








TTTCTGAGAG







 


13531-R
TCCCTCTGCCCCTCACCTGCTGCTTGGGGTTTGGGGTGCAGACATTGCCAGAGG
9
131671687
131671787
1
451



ATGGGCAGCAGACTGACCTTCAACCCCACAGGTATCCACCACAGTGG







 


13627-L
CCCACCATCCTAGCTTGCCTGAGACTGTCCTGGTTTTAGCACTGAAAGTGCCTG
2
71607460
71607555
1
452



TTCCAGGAAACCCTGCAGTCTCCAGCAAACTGGGACAGTGGG







 


13628-L
GCTTCTCTGAGGATGAAAGACCCATTGAGGAGAAGGTTCTGCTGGCTGAGAACC
22
48323039
48323125
−1
453



TTCCTCTCCATGGGTCTTTCATCCTCAAAGAAC







 


13629-R
GCTCTGTGATTGCCTCTGATCAGGCAAAATTGCAGACTGTCTTCCCAAATAGCC
2
102415176
102415263
1
454



TGCAACTTTGCCTGATCAGAGGCAGTCACAGAGC







 


13629-L
GCTCTGTGATTGCCTCTGATCAGGCAAAATTGCAGACTGTCTTCCCAAATAGCC
2
102415176
102415263
1
455



TGCAACTTTGCCTGATCAGAGGCAGTCACAGAGC







 


13630-L
ATTCTCTTAGGTTGGTGCAAAAGTAGTTGTGGTTTTGCCATTCATTTCAGTGAT
2
126329376
126329466
1
456



AAAAACCGCAATTACTTTTGCACCAACCTAATCGAAT







 


13640-L
TTTAAGAACTGGATATGATGACTGAAATAAGCTCCATATCAATGAGAATTTCAA
3
19331342
19331429
−1
457



TGGGATTATGTGCAGTCAATGTCCAGTAATTAGA







 


13642-R
TTAGTTTGGTGCAAAAGTAATCACGGTTTTTGCTATTGAAAGTAATAGCAAAAA
3
38039376
38039471
1
458



CTTTCAATAGCAAAAACTGTGATTACTTTTGCATCAATCTAA







 


13655-L
AGAAAAATGTTAGGGTGGTGCAAAAGTGATCGTGGTTTTTGCAATTTTTTAATG
4
174425875
174425970
1
459



ACAAAAACCACAATTACTTTTGCACCAACCTAACCTTGTTTT







 


13661-R
AGTTTCCATGATAGGGAAACCAGGCAAGAAATATTGTCTCCTCAAGTTGCGACG
6
2799256
2799348
−1
460



AGACAGTAGTTCTTGCCTGGTTTCTCTATCATGGAGTCT







 


13665-R
TGTGCTCCAGTACATATAAAGAGACTTATTAAGATGATCTTTTCTTAATAAGTC
6
82532186
82532261
−1
461



TCTTTATATGTACTGGAGCCCG







 


13667-R
TATTGGGTGGGTGCAAAAGTAATTGCGGTTTTTGCTATTAGTTTCAATGGTAAA
6
108204187
108204270
−1
462



AACCGTGACTACTTCTGCACCAACCTAGTA







 


13670-L
AAAATAGGACTTTTGAAGGAAGAGTTTTTTTTCACATTTTCACACTTTTCCTTC
6
135559044
135559111
1
463



AAAAGTCATATTTT







 


13672-R
AGAGAAGTTTTCAGTGTAACTCAACATTTGAAGTGTACTTGCCCTTGGACCAAG
6
154546929
154547022
−1
464



CAATTCTTCAAATGTTGAGATACACTGAAAACTTTCTCT







 


13677-L
GGATAATAGGACAAGACAAATTACAGATTGTCTCAGAGAAAACAAATGAGTTAC
7
138379372
138379461
−1
465



TCTCTCGGACAAGCTGTAGGTCCTACCTAAATGTCC







 


13677-R
GGATAATAGGACAAGACAAATTACAGATTGTCTCAGAGAAAACAAATGAGTTAC
7
138379372
138379461
−1
466



TCTCTCGGACAAGCTGTAGGTCCTACCTAAATGTCC







 


13679-L
TAGGTTCATGCAAAAGTAGTTGTGGTTTTGCCATTACTTTCAATGGATGGCAAA
8
69201988
69202068
−1
467



AACAGCAATTACTTTTGCACCAACCTA







 


13681-R
GAGGGAAAGCAGGCCAACCTCGAGGATCTCCCCAGCCTTGGCGTTCAGGTGCTG
8
96154315
96154400
−1
468



AGGAGATCGTCGAGGTTGGCCTGCTTCCCCTC







 


13686-L
AAGGAACAGGGGACACTTGTAATGGAGAACACTAAGCTATGGACTGCTATGGAC
9
20401145
20401236
−1
469



TGCTAGTGCTCTCCGTTACAAGTATCCCCTGTTACCTT







 


13687-R
GATATTTGAACCTCCTCCCGTGAATCACAAATGTCCTTAATAGCAATCCTTAAA
9
67905119
67905217
1
470



TGCCATTAAGGACATTTGTGATTGATGGGAGGAGGATGAAATATT







 


13691-R
GGGAAAAAAAAAAGGATTTGTCTTGTAGCCAGGATATTGTTTTAAAGAAAATCC
9
113734202
113734277
1
471



TTTTTGTTTTTCCAGGTGGACC







 


13692-R
AGGGCTGCTAGATTTAATGGATCAAATCACAAGATGCCTAGTTAAATTTGAATT
9
122531275
122531369
−1
472



TTAAATTTAACTGGACATCTTGCATTTTATCTGGTAATCCT







 


13694-L
CTCTGCCCCATCTCCACCTGGACCCAGCGTAGACAAAGAGGTGTTTCTACTCCA
X
1372805
1372890
1
473



TATCTACCTGGACCCAGTGTAGATGGGAGGAG







 


13694-R
CTCTGCCCCATCTCCACCTGGACCCAGCGTAGACAAAGAGGTGTTTCTACTCCA
X
1372805
1372890
1
474



TATCTACCTGGACCCAGTGTAGATGGGAGGAG







 


13696-L
GTAGGATAAATGACTCATCCTAGCTTGCCTGAGACTGTCCCAGTTTGAAAACTG
X
7115401
7115497
1
475



GACCTCATCAGTCCTAGACACACTGGGATGTGGTTCACCCTAT







 


13704-R
TTTATTGTGAAATATGTCATTAATATGTACTGACAAAGCGTATCTGTGTAATAA
10
5769057
5769155
1
476



ATATGCTTTTTGTCAGTACATGTTAATGGTATATTTCATAACAAA







 


13705-L
CTGTTCCGGGCATCACCTCCCACTGCAGAGCCTGGGGAGCCGGACAGCTCCCTT
10
6234161
6234250
1
477



CCCAGGCTCTGCAGTGGGAACTGATGCCTGGAACAG







 


13709-R
GATCATTATTCAGGCCGGTCCTGCAGAGAGGAAGCCCTTCTGCTTACAGGTATT
10
103351157
103351253
1
478



GGAAGGGCTTCCTCTCTGCAGCACCGGCCTGAATAATGTAATC







 


13710-R
CAGTTTCTCTTCCATCCCATATCTTAACAGCTAATCTAGTAAATTCTATCTTCA
11
33155668
33155761
1
479



GAAGATTTGCTGTTAAGATATGGGATGGAGGAGAAATCTG







 


13716-L
TTTTTCTCCCAGTCAAGAGTTACTAGAACTATTCAACCTTCAGCTGTGTTGAAT
12
21562856
21562939
1
480



AGTTTTAGTAACTCTTGACTGGGAGAAAAG







 


13716-R
TTTTTCTCCCAGTCAACAGTTACTAGAACTATTCAACCTTCAGCTGTGTTGAAT
12
21562856
21562939
1
481



AGTTTTAGTAACTCTTGACTGGGAGAAAAG







 


13717-L
AGCTGGACTGAGAGAAATTTATTCTTGGTAGGTTGTACATTCCTAAACATGTAC
12
29307836
29307923
1
482



AACGTACTAAGAATAAATTTCTCTCAGTCCAGCT







 


13717-R
AGCTGGACTGAGAGAAATTTATTCTTGGTAGGTTGTACATTCCCAAACATGTAC
12
29307836
29307923
1
483



AACGTACTAAGAATAAATTTCTCTCAGTCCAGCT







 


13719-L
GGTTAGCACAGAGTGGGAGCTCTAGAAAGATTGTTGACCAATCATCTTATTGAC
12
64931121
64931211
1
484



TAGACCATCTTTCTAGAGTATAACTATTTTGGACACC







 


13721-R
GCTGCGTTTGCACTGCTTCTCCAAAACCACATTATAGGTACTAAACAACATTGT
12
70365967
70366062
−1
485



TTAGTACCTATAATGTGCTAGACTCCTGGCTGCTAGCGAGGT







 


13727-R
GGTATGTATCTGCTTTTCCGGAGTTGTAAGTGTTGACAATATCCAGAATGACAT
14
90236780
90236877
−1
486



TGTCTTTGTCAACACTTACAACTCTGGAAAAGCAGATACATACC







 


13727-L
GGTATGTATCTGCTTTTCCGGAGTTGTAAGTGTTGACAATATCCAGAATGACAT
14
90236780
90236877
−1
487



TGTCTTTGTCAACACTTACAACTCTGGAAAAGCAGATACATACC







 


13729-L
AGTATGACACCTCAAAGAAGCAATACTGTTACCTGAAATAGGCTGCGAAGATAA
15
33451756
33451852
−1
488



CAGTATTTCAGATAACAGTATTACATCTTTGAAGTGTCATATT







 


13731-L
AAAAAAAGGGAAAGAAGAACTGTTGCATTTGCCCTGCACTCAGTTTGCACAGGG
15
63798639
63799723
−1
489



TAAATGCAATAGTTCTTCTTTCCCTTTTTTT







 


13734-L
GTTTTGGATTTTGGACTTTTTCAGATTTGGGGATATTTGCATTATACTTATCCT
15
88228177
88228256
−1
490



AAATCTGAAAGTCCAAAACCTGAAAT







 


13745-L
GCCTCAAATTTAAGGAGGGACTCACTCACAGGATTGTGCAAATGCAAAGTTGGC
16
29517998
29518092
−1
491



TTTTGCATGACCCTGGGAGTAGGTGCCTCCTTAAATTTTGC







 


13748-L
GCCTCAAATTTAAGGAGGGACTCACTCACAGGATTGTGCAAATGCAAAGTTGGC
16
21424868
21424962
−1
492



TTTTGCATGACCCTGGGAGTAGGTGCCTCCTTAAATTTTGC







 


13753-L
CTGTAGGTTCTGTCTTGGGCCACTTGGATCTGAAGGCTGCCCCTTTGCTCTCTG
17
52323626
52323720
−1
493



GGGTAGCCTTCAGATCTTGGTGTTTTGAATTCTTACTATAG







 


13755-L
TGCCTACAGTGAATCCCTAGTGGTCAGAGGGCTTATGATATATTGTGAGAGCCA
18
3875346
3875439
1
494



TGTCATAAGCCTTTTGGCCACTAGGGATTCAATGTATGCA







 


13761-L
TTGAGGAAGGGTCAGGCATGAGGAGGATCTGAAGGATTGGACTCAGGTTCGAAA
19
60428648
60428739
−1
495



CCTCCACTTCCTCCTCATCTCCTACCCTCTCCACTCAG







 


14069-R
AGTATAGTGGTGAGTATCCCCGCCTGTCACGCGGGAGACCGGGGTTCGATTCCC
12
123990092
123990209
−1
496



CGACGGGGAGGCCGGGTACTTTCGTATTTTTAAATACAGAGGGGAGACTTTGTT








GGCGATGCTT







 


14085-L
GGCCTAATGGATAAGGCATTGGCCTCCTAAGCCAGGGATTGTGGGTTCGAGTCC
16
3142910
3143008
1
497



CACCCGGGGTAAAGAAAGGCCGAATTTTAGTGTTCCTTATCGGGC







 


14086-L
GAAGGCAGCTACACATGCGTGGCGGTTCCATGGTGTAATGGTTAGCACTCTGGA
15
63948435
63948548
−1
498



CTCTGAATCCAGCGATCCGAGTTCAAATCTCGGTGGAACCTGCATTGGTTTTTG








TTTTTT







 


14093-L
TCAGTGGTAGAGCATTTGACTGCAGATCAAGAGGTCCCCGGTTCAAATCCGGGT
17
34564239
34564330
−1
499



GCCCCCTCTGTGCTCCGGAGTTACCTCGTTTTGTTGGT







 


14111-L
TGTAGTGTTTCTTACTTTAAATATGTAAAATGTGTAACATGCAGAGCGAAAGGG
Y
8054989
8055049
−1
500



GCAGTGA







 


14113-R
AAAGTCGGTGCCGGAGGCTCCCAGCTCAGATCGCCGAAGCGTCGGACTACCGTT
19
2427098
2427192
1
501



GGTTTCCGCAACTTCCTGGATTATCCTCGCCAAGGACTTTG







 


14113-L
AAAGTCGGTGCCGGAGGCTCCCAGCTCAGATCGCCGAAGCGTCGGACTACCGTT
19
2427098
2427192
1
502



GGTTTCCGCAACTTCCTGGATTATCCTCGCCAAGGACTTTG







 


14117-R
AATCAACTTCCTAGGCACACTTAAAGTTATAGCTACATCAGTTATAACTATATC
11
46731242
46731329
−1
503



AGTTAAAACTTTAAGTGTGCCTAGGAAGTTGATT







 


14121-R
ACATATTGGTCTGTTGTCAGGCTCAGCAGCATGGCTGACTAAAGACATTGACAT
1
229467154
229467262
1
504



CATGACATTGTCATGTTGCTCTGCTGTTGCCCCTGGCTCTAGCTGGCCTACT








TGT







 


14137-R
AGCCCTGGGGGTGGTCTCTAGCCAAGGCTCTGGGGTCTCACCCTTGGCTGGTCT
8
27346804
27346872
1
505



CTGCTCCGCAGGCCT







 


14151-R
AGTGGAGAAGGCCACGATTTTTTTGATGTCATTTTGTGTAAGGGCGCAGACTGC
5
134289131
134289250
1
506



TGCGAACAGAGTGGTGATAGCGCCTAGGCATAGTGTGAGAGTTTGGATTAGTGG








GTTATTCTCTGC







 


14154-L
ATAGTTTTAGAGTTTCTAAGGATCATGTCTGTGAGTCAGGATTCCAGAGACCAT
13
60110163
60110256
−1
507



GGTCCTGATGGGATGGAGCCTGGAGACGTCATTGAGAGTA







 


14156-R
ATGATGGCTATGGATTTGGGTCAGATAGATTTGGAAGAGGTAAGGTAAGAATTG
5
178977520
178977634
−1
508



AATTTCTCAGTTGAAGGATGCTTACACTCTTGTCCATCTAGACCTCAATTACTG








TTTTTCA







 


14161-R
ATTCTAGATTAAATGGTCAAGGAAGACAGTCTGTGGCAAAGGCCCAGTTCAAGT
10
122640045
122640164
−1
509



TTAGAACTGAACAGCCTCTGGCAATCTATCTTCCACAAATAGGCAGCATTTTAA








AGGTCTCAGAGA







 


14170-L
CACGTCACCTGATATCAGGTATTTACTCTGAACTAGCTCTGTGGATCCTGACAG
8
92286868
92286982
1
510



ACAGCCTGATAGACAGGATCCACAGAGCTAGTCCAGAGTAAAAGACCTAAATCA








GCTGTGG







 


14177-L
CAGTAGTGGTTAGTACTCTGAATTAACTTATCTAAATCAATGAAAATAATAGTG
1
69384134
69384197
−1
511



AACCTCTGCT







 


14200-L
CCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCAGGAGCCCCCGTTGCCAC
22
29111449
29111549
−1
512



TGCTGCTGCTGCTGCCCCTGCTGCCACTGTGGCTGGGCCTGGCAGGG







 


14220-L
CGGATAATTGTAGCACTTCCTGGTTCATACGGATAATTGTAGCACTTCCTGGTT
X
2557830
2557935
1
513



TGCAGGGACATTGCAATACTTCCTGGTTCATATGGATAATTGTAGCACTTCT







 


14226-R
CTCAAACAACTCAATAGGAAAAAAAACTAATAATCTGATTTAAAAACGGGCAAA
10
93886209
93886290
1
514



AGAGAGTTTCTAAGGATCATGTCTGAGA







 


14229-R
CTCGGCCTCCCTTCATGGTGGGACCAGGCCCAGCAGGGAATGTCAGGGCCACCC
7
44057873
44057967
1
515



CTGACCTTCACTGTGACTCTGCTGCAGAGGGTGGCCTGGAG







 


14244-L
CTGTGCTTTGTGTGTTGGAGGATGAAAGTACGGAGTGATCCATCGGCTAAGTGT
16
70349796
70349899
−1
516



CTTGTCACAATGCTGACACTCAAACTGCTGACAGCACACGTTTTTCACAG







 


14244-R
CTGTGCTTTGTGTGTTGGAGGATGAAAGTACGGAGTGATCCATCGGCTAAGTGT
16
70349796
70349899
−1
517



CTTGTCACAATGCTGACACTCAAACTGCTGACAGCACACGTTTTTCACAG







 


14275-L
GAAACTCGGTGACTCCAGGGACTGCCTTAGGAGAAAGTTTCTGGAAGTTCTGAC
22
26646503
26646607
−1
518



ATTCCAGAAACTTTCTCCTAAGGCAGTCCCTGGGAGTCACTGAGTCAGTCC







 


14288-R
GCAACTCGCCTGGCTCCCTTCTCTCCGTCTGCCTCCTGGCCGCGGGGCCCGGAG
6
41809263
41809336
−1
519



AGCTGGGAGCCAGAGGGTGT







 


14289-R
GCAAGGGTTGAGGAGGGACTTAACATCAATGAATTCATGAGGTGATGTAGTCTC
20
33683569
33683634
−1
520


 CCCTACCTAGGT








14303-R
GCCCTTCGGTAGCTGGTCCCTTAACTCAGTGGTGAATGGCGACCGGATGGAGCT
16
74108610
74108689
−1
521



CTAGGGAAGCGACAGCAGCGGCGGGT







 


14327-R
GGAAGAGGGCTTAGGTGCACGCTCTAGCGGGGATTCCATATTGGGCCAATTCCC
1
54292328
54292434
1
522



CCAATGTTGGAATCCTCGCTAGAGCGTGCACTTCTGGAAGCTAGGAACCTCC







 


14328-R
GGAAGAGTCAAGTCAAGGCCAGAGGTCCCACAGCAGGGCTGGAAAGCACACCTG
12
103509541
103509624
1
523



TGGGACTTCTGGCCTTGACTTGACTCTTTC







 


14332-L
GGACTGACTCAGTGACTCCCAGGGACTGCCTTAGGAGAAAGTTTCTGGAATGTC
22
26646503
26646611
1
524



AGAACTTCCAGAAACTTTCTCCTAAGGCAGTCCCTGGAGTCACCGAGTTTCT








CTT







 


14336-R
GGCACAGACTCATCCCTGATCAAAGCCTCCACCCCCCCCCCAAAAAAAAGATCT
19
57737967
57738064
−1
525



GGGCACCTACTTGGGAAGCTGAGGCAGGAGAATCACTTGAACCC







 


14338-R
GGCATGGAGTCCTGTGGCATCCACGAAACTACCTTCAACTCCATCATGAAGTGT
7
5534260
5534343
−1
526



GACGTGGACATCCGCAAAGACCTGTACGCC







 


14347-R
GGGCAGGAGGGAGTGGGGTGGGACCCAGCTGTTGGCCATGGCGACAACACCTGG
1
226651366
226651439
−1
527



GTTGTCCCCTCTAGGGTCCA







 


14361-L
GGGGCTAATCGCCTGGACTGCCTGGAGAAAGCGAAGCTAGTACCCCCTTTCTCC
17
53514138
53514215
−1
528



AACAGTCCTTAGTTTCCAGGCCCT







 


14367-R
GGTAATCTCATCAGAAAGATAGGCAGCTTCCAAGTCCCAGGGCCTCGTAAGCAG
2
135573962
135574057
1
529



AGGCACAGTTATGGATGATGATATCTGAGTGATATTGTGCTT







 


14371-R
GGTTAACTTACAATAAATGAAGCTTTTATGCAGGTTTCCGTAGCGTAGTGGTTA
9
103368541
103368597
−1
530



TCA







 


14375-R
GTAAGGTGGAGAGATTACCGTGTTATAAAGAACTTTGGGATATTTTTCAAAATT
3
12602278
12602397
−1
531



AACCTGACCATTCTTTTGAAACCAGAGTCCTTAACAAGCATTGAGATATATTTC








TCCATGAAGGCT







 


14380-R
GTGAGAAATGATGAGGGTCAACATTCTTCATACCAAAGTGAAGACATGAGATCC
3
52697944
52698012
1
532



AACTCTGAGCTCACC







 


14385-L
GTGGCCGAGGACTTTGATTGTACATTGTTCTTTTTTTAATAGTCATTCCAAATA
6
146967008
146967109
1
533



TCATGAGATGCATTGTTACAGGAAGTCCCTTGCCCTCCTAAAAGCCAC







 


14390-L
GTTGCTATCGGGGACTACAATGGCCACGTCGGTCTGGGTGTTAAGTGCTCCAAG
1
51489239
51489358
−1
534



GAGGTGGCCACCGCCATCCATGGGGCCATCATCCTGGCCAAGCTCTCCATTGTC








CCCGTGCGCAGA







 


14399-L
TCAAGGAAAAGCTGTGCTACGTCGCCCTGGACTTCGAGCAGGAGATGGCCACGG
9
102533969
102534043
1
535



CAGCCTCCAGCTCCTCCCTGG







 


14409-R
TCCATCCGTATTGAGGATGAGGACACCTCTGAACTCACAGAGCAGGCTGTGAGT
X
47580920
47581003
−1
536



TTAGAGCTGTCTGCTCTAAACTCAGGTGGA







 


14415-R
TCTATTGCTATGCCTCCAAGTTCATTAATATTTTCTTCTCATTGTAAAGACTGG
11
32063048
32063157
1
537



GGGACCTCTTTCTTAAGTGATCTTTGTCTTAAGATTTGGTGCAATATATCAGT








AGA







 


14442-R
TGTGCTTGCGCATAACTGGGGCCGCCTGGCCTCCCGCGGGCGGCCTTTTTAACC
17
59577497
59577560
1
538



GCGAGCGACA







 


14452-L
TTCCTTAACTAAAGTACTCCCTCTCAAATCAAAGTGGTTATGGTTCTAAAAACT
4
128346613
128346694
1
539



CCGATTGGAGAATTTACAGGATAGGAGG







 


14454-R
TTGGGCATGGAATCCTGCGGCATCCACAAGACCACCTTCAACTCCATCATGAAG
X
53188939
53189027
−1
540



TGTGACGTGGACATCCGCAAAGACCTGTACGCCAA







 


14469-R
CTGGTGGCTGGGCCGACGACTCGGGGCGCGGCCGTGAGCGCAGAGGCCATGGAG
7
101730795
101730904
1
541



CCGGAGCTCGCGGACACCTCTGCACTCAAGGCGGCGCCCTACGGCCACTCGCG








GAG







 


14479-R
GAGAATTTGTATATTTTCTATTTAGAAGCCTATAGATAGAATATAGGAAAAAAA
2
69512355
69512457
−1
542



GCTATATTTTTCTTATAGGCTTCTAAGTAGAAAATGTATTTGCAAGGAT







 


14482-R
GATCTATGACGTGGAGCTAACCAAACTAATATACCCATATTCTGGCTAGGTGAT
18
35510661
35510764
1
543



CATCAGAATATGGGTATATTAGTTTGGTTAGCTCTACATTGTAGATCTAT







 


14483-R
GATGGCTTTTATCATTGGGCCAGTGACGACCCATTCAGCCGTATCAGTGAAGAG
16
76971867
76971976
1
544



TGAAGCACTGCACTCTTTAAGGATAGGGCTGAATGGTTTATCACCCATTCAGC








GGT







 


14485-L
GCACCACTAGACTCCAGCCTGGGCAAGAAAGTGAGACTCTGCCTCTTTTTTGTT
7
74097020
74097123
−1
545



TCTGAGCCAGAGTCTTGCTCTGTTGCTCAGGCTGGAGTGCAATGGCGTGA







 


14494-L
GCGCGGCCGCGGGAGGTGTAACAGGACTGGACTCCCGGCAGCCCCAGGGCAGGG
15
81527083
81527169
1
546



GCGTGGGGAGCTGGTCCTAGCTCAGCGCTCCCG







 


14499-L
GGACGTAGTGGTGTAGAGAGGCATTAAGAACGCAGGTGGCAGGGCCAGCCACCA
20
49502833
49502954
−1
547



GGAGGGCTGCGTGCCACCCGGGCAGCTCTGCTGCTCACTGGCAGTGTCACCTGC








GGAAACTCTCCATC







 


14508-L
GTAGCAGTGGTGGTGGTAGCAAGGGGGTGATGTAGATTCTGAAGAGCAGCCCTT
17
40367490
40367629
−1
548



CCTGTTAGGGGAAAAGTCCTGATCCGGGAACCCACAGCCCCGTTCCTGGGCTTC








TCCTCTGTAGCCAGCCTCAGCCGAGCAGCTGC







 


14527-R
TCCCAATACGAGGCACTGAGTTAAGTGTTCTACACAGATAATCTGTTATTAATG
11
17982809
17982918
−1
549



AGAGAATAGAATAGACAATCTGTGTAGAGTGCTTAACCTGGTGCCCAGTGTTT








TCA







 


14532-L
TGCGTTGGCCTGTGGGCATGGCCTGAGACCAGGACTGGATGCACCACTCTCCCT
2
240531091
240531197
−1
550



GTGATGAGGTGAAGCCAGCTCTGGTCTGGGCCATTTCACAGGATTCCAGAAGC







 


14539-R
TTAAAGACCTTAAGAGGCAACAAAGACTATACTTTCAGGGATCATTTCTCTAGT
4
159841271
159841378
1
551



TCAATACTACAGAAGTTTCTCTGAAGGTGTAGCAAGCACCAGAAACCACGAGGA







 


14541-R
TTAATATTTCATCCTCCTCCCATCAATCACAAATGTCCTTAATGGCATTTAAGG
9
67905114
67905219
−1
552



ATTGCTATTAAGGACATTTGTGATTCACGGGAGGAGGTTCAAATATCATGAA







 


14543-L
TTATATCCAGAGCCTGAATGAAAGAGCCAGTGGTGAGACAGTGAGTTGATTACT
10
74150779
74150880
1
553



TCTCACTGTTTCACCACTGGCTCTTTGGTTCATGCTAACAATGTATCT







 


14549-R
TTGTAACATCCGGTGTGTTGTAACATTCCAGTGTGTTGTAACATTTGGTGTTAC
12
131953101
131953206
−1
554



ATGCTGTGTTGTAACATTCTGGTGTGTTGTAACATTCCCGGTGTGTTGTAAC







 


14556-R
AAATACAAAATAATTCGAGAATAAAGACTATGCTTTCAGGGATCATTTCTATAG
10
88799960
88800071
1
555



TTCGTTACTCGGGAAGTTTCTCTGAACGTGTAAAGCACCGAACAAAAAAAAAAA








AAAC







 


14567-R
ACACATTTTCTTTGCTAAGTCCCTTCTTTCTATCCTAGTATAACTTGAAGAATT
7
19711490
19711597
−1
556



CAAATAGTCATGCTAGGATAGAAAGAATGGGACTTGGCCAGGGAAGAAGAGTTG







 


14570-R
AGAAATTATGCCACTTTTAATTTCAGCTACTACCTCTATTAGGATTTGGGAGTT
6
35740457
35740557
−1
557



ATACTAATAGAGGTAATAGTTGAAATTAAGAGTGGATGAGTTCTGGT







 


14575-R
AGCTGATTACATTATTCAGGCCGGTCCTGCAGAGAGGAAGCCCTTCCAATACCT
10
103351150
103351257
−1
558



GTAAGCAGAAGGGCTTCCTCTCTGCAGGACCGGCCTGAATAATGATCCTAACGC







 


14578-R
AGTAACATCCAGATGTGTTGTAACATTCCAGTGTGTTGTAACATTCCTGTAACA
12
131953328
131953435
−1
559



TTCCAATGTGTTGTAACATTCTGGTGTGTTGTAACATTCCTGTAACATTCTGGT







 


14581-L
ATACCATTGGGCCTTGCTTCTTTATCCAGCTTGTTACTATATGCTTTTTAAATG
14
59183411
59183513
−1
560



GGGCACAGAGTGACAAGCTGGTTAAAGAAGCAAGACCCCTTCAAGATTA







 


14583-L
ATGGGGGAACCACAGGCAGCAAATGGCCAGAGCTCACACAGAGGGATGAGTGCA
2
111795045
111795152
−1
561



CTTCACCTGCAGTGTGACTCAGCAGGCCAACAGATGCTATCAGGGAAGAGCACT







 


14584-L
ATGGGGGAACCACAGGCAGCAAATGGCCAGAGCTCACACAGAGGGATGAGTGCA
2
87710376
87710483
1
562



CTTCACCTGCAGTGTGACTCAGCAGGCCAACAGATGCTATCAGGGAAGAGCACT







 


14596-R
CCAACCCCATCTTCTTAAATGTCTTACTGCTTTTACTGTTCCCTCCTAGAGTCC
2
86273648
86273757
−1
563



ATTCTTTACTCTAGGAGGGAATAGTAAAAGCAGTAAGACATTTAGTAAAAGGC








TTT







 


14601-L
CCCAGGTAAAGGGCCCAGGTGACCCGGGGCTGCCCTGAACGGGCCCGGCTCTGG
11
63838955
63839070
1
564



TGCGCTTGCTCAGCCAGGCCCGCTCCCCGCTGCCCCCTAGGCTTCTCATCGCTG








TCGCTGTC









A similar analysis was carried out to identify microRNAs in miRBase (human version 14.0) that are at increased levels in the sepsis patient sample as compared to the 77 non-sepsis patient smRNASeq datasets. Again, the microRNAs were ranked from 1 to 5, depending on whether the sepsis patient smRNASeq dataset contained the highest number of counts of the particular microRNA, the second highest, etc. The results are shown in Table 14.







TABLE 14







microRNAs from miRBase found at higher


numbers in a sepsis smRNASeq dataset










microRNA

microRNA
SEQ ID


name
RANK
Sequence 5′->3′
NO













miR-101
1
TACAGTACTGTGATAACTGAA
565


 


miR-140-5p
1
CAGTGGTTTTACCCTATGGTAG
566


 


miR-142-3p
1
TGTAGTGTTTCCTACTTTATGGA
567


 


miR-142-5p
1
CATAAAGTAGAAAGCACTACT
568


 


miR-143*
1
GGTGCAGTGCTGCATCTCTGGT
569


 


miR-144
1
TACAGTATAGATGATGTACT
570


 


miR-144*
1
GGATATCATCATATACTGTAAG
571


 


miR-148a
1
TCAGTGCACTACAGAACTTTGT
572


 


miR-1537
1
AAAACCGTCTAGTTACAGTTGT
573


 


miR-15a
1
TAGCAGCACATAATGGTTTGTG
574


 


miR-16-1*
1
CCAGTATTAACTGTGCTGCTGA
575


 


miR-16-2*
1
CCAATATTACTGTGCTGCTTTA
576


 


miR-185*
1
AGGGGCTGGCTTTCCTCTGGTC
577


 


miR-2115
1
AGCTTCCATGACTCCTGATGGA
578


 


miR-2115*
1
CATCAGAATTCATGGAGGCTAG
579


 


miR-223
1
TGTCAGTTTGTCAAATACCCCA
580


 


miR-223*
1
CGTGTATTTGACAAGCTGAGTT
581


 


miR-27a*
1
AGGGCTTAGCTGCTTGTGAGCA
582


 


miR-30e
1
TGTAAACATCCTTGACTGGAAG
583


 


miR-30e*
1
CTTTCAGTCGGATGTTTACAGC
584


 


miR-338-3p
1
TCCAGCATCAGTGATTTTGTTG
585


 


miR-338-5p
1
AACAATATCCTGGTGCTGAGTG
586


 


miR-374a*
1
CTTATCAGATTGTATTGTAATT
587


 


miR-451
1
AAACCGTTACCATTACTGAGTT
588


 


miR-486-5p
1
TCCTGTACTGAGCTGCCCCGAG
589


 


miR-548e
1
AAAAACTGAGACTACTTTTGCA
590


 


miR-548f
1
AAAAACTGTAATTACTTTT
591


 


miR-548j
1
AAAAGTAATTGCGGTCTTTGGT
592


 


miR-548n
1
CAAAAGTAATTGTGGATTTTGT
593


 


miR-548q
1
GCTGGTGCAAAAGTAATGGCGG
594


 


miR-576-3p
1
AAGATGTGGAAAAATTGGAATC
595


 


miR-582-3p
1
TAACTGGTTGAACAACTGAACC
596


 


miR-617
1
AGACTTCCCATTTGAAGGTGGC
597


 


miR-618
1
AAACTCTACTTGTCCTTCTGAGT
598


 


miR-624*
1
TAGTACCAGTACCTTGTGTTCA
599


 


miR-625*
1
GACTATAGAACTTTCCCCCTCA
600


 


miR-627
1
GTGAGTCTCTAAGAAAAGAGGA
601


 


miR-628-5p
1
ATGCTGACATATTTACTAGAGG
602


 


miR-629*
1
GTTCTCCCAACGTAAGCCCAGC
603


 


miR-640
1
ATGATCCAGGAACCTGCCTCT
604


 


miR-1225-5p
2
GTGGGTACGGCCCAGTGGGGGG
605


 


miR-1255a
2
AGGATGAGCAAAGAAAGTAGATT
606


 


miR-126*
2
CATTATTACTTTTGGTACGCG
607


 


miR-148a*
2
AAAGTTCTGAGACACTCCGACT
608


 


miR-26b*
2
CCTGTTCTCCATTACTTGGCTC
609


 


miR-30a*
2
CTTTCAGTCGGATGTTTGCAGC
610


 


miR-425
2
AATGACACGATCACTCCCGTTGA
611


 


miR-450b-5p
2
TTTTGCAATATGTTCCTGAATA
612


 


miR-503
2
TAGCAGCGGGAACAGTTCTGCAG
613


 


miR-514
2
ATTGACACTTCTGTGAGTAGA
614


 


miR-548b-5p
2
AAAAGTAATTGTGGTTTTGGCC
615


 


miR-548d-3p
2
CAAAAACCACAGTTTCTTTTGC
616


 


miR-548d-5p
2
AAAAGTAATTGTGGTTTTTGCC
617


 


miR-580
2
TTGAGAATGATGAATCATTAGG
618


 


miR-616*
2
ACTCAAAACCCTTCAGTGACTT
619


 


miR-1179
3
AAGCATTCTTTCATTGGTTGG
620


 


miR-1244
3
AAGTAGTTGGTTTGTATGAGATGGTT
621


 


miR-126
3
TCGTACCGTGAGTAATAATGCG
622


 


miR-1291
3
TGGCCCTGACTGAAGACCAGCAGT
623


 


miR-140-3p
3
TACCACAGGGTAGAACCACGG
624


 


miR-145*
3
GGATTCCTGGAAATACTGTTCT
625


 


miR-146a
3
TGAGAACTGAATTCCATGGGTT
626


 


miR-146a*
3
CCTCTGAAATTCAGTTCTTCAG
627


 


miR-146b-3p
3
TGCCCTGTGGACTCAGTTCTGG
628


 


miR-146b-5p
3
TGAGAACTGAATTCCATAGGCT
629


 


miR-148b*
3
AAGTTCTGTTATACACTCAGGC
630


 


miR-150*
3
CTGGTACAGGCCTGGGGGACAG
631


 


miR-17*
3
ACTGCAGTGAAGGCACTTGTAG
632


 


miR-181a*
3
ACCATCGACCGTTGATTGTACC
633


 


miR-191*
3
GCTGCGCTTGGATTTCGTCCCC
634


 


miR-199b-5p
3
CCCAGTGTTTAGACTATCTGTTC
635


 


miR-26a-1*
3
CCTATTCTTGGTTACTTGCACG
636


 


miR-29b-2*
3
CTGGTTTCACATGGTGGCTTAG
637


 


miR-340*
3
TCCGTCTCAGTTACTTTATAGC
638


 


miR-361-3p
3
TCCCCCAGGTGTGATTCTGATTT
639


 


miR-422a
3
ACTGGACTTAGGGTCAGAAGGC
640


 


miR-425*
3
ATCGGGAATGTCGTGTCCGCCC
641


 


miR-454*
3
ACCCTATCAATATTGTCTCTGC
642


 


miR-502-5p
3
ATCCTTGCTATCTGGGTGCTA
643


 


miR-542-3p
3
TGTGACAGATTGATAACTGAAA
644


 


miR-550
3
AGTGCCTGAGGGAGTAAGAGCCC
645


 


miR-551a
3
GCGACCCACTCTTGGTTTCCA
646


 


miR-573
3
CTGAAGTGATGTGTAACTGATCAG
647


 


miR-598
3
TACGTCATCGTTGTCATCGTCA
648


 


miR-643
3
ACTTGTATGCTAGCTCAGGTAG
649


 


miR-671-5p
3
AGGAAGCCCTGGAGGGGCTGGAG
650


 


let-7f
4
TGAGGTAGTAGATTGTATAGTT
651


 


let-7g
4
TGAGGTAGTAGTTTGTACAGTT
652


 


miR-103
4
AGCAGCATTGTACAGGGCTATGA
653


 


miR-103-2*
4
AGCTTCTTTACAGTGCTGCCTTG
654


 


miR-107
4
AGCAGCATTGTACAGGGCTATCA
655


 


miR-1197
4
TAGGACACATGGTCTACTTCT
656


 


miR-1250
4
ACGGTGCTGGATGTGGCCTTT
657


 


miR-1277
4
TACGTAGATATATATGTATTTT
658


 


miR-1278
4
TAGTACTGTGCATATCATCTAT
659


 


miR-1299
4
TTCTGGAATTCTGTGTGAGGGA
660


 


miR-143
4
TGAGATGAAGCACTGTAGCTC
661


 


miR-150
4
TCTCCCAACCCTTGTACCAGTG
662


 


miR-155*
4
CTCCTACATATTAGCATTAACA
663


 


miR-181a
4
AACATTCAACGCTGTCGGTGAGT
664


 


miR-181b
4
AACATTCATTGCTGTCGGTGGGT
665


 


miR-186
4
CAAAGAATTCTCCTTTTGGGCT
666


 


miR-196b
4
TAGGTAGTTTCCTGTTGTTGGG
667


 


miR-1976
4
CCTCCTGCCCTCCTTGCTGT
668


 


miR-21
4
TAGCTTATCAGACTGATGTTGA
669


 


miR-219-1-3p
4
AGAGTTGAGTCTGGACGTCCCG
670


 


miR-221*
4
ACCTGGCATACAATGTAGATTT
671


 


miR-378
4
ACTGGACTTGGAGTCAGAAGG
672


 


miR-423-3p
4
AGCTCGGTCTGAGGCCCCTCAGT
673


 


miR-500*
4
ATGCACCTGGGCAAGGATTCTG
674


 


miR-502-3p
4
AATGCACCTGGGCAAGGATTCA
675


 


miR-532-5p
4
CATGCCTTGAGTGTAGGACCGT
676


 


miR-548k
4
AAAAGTACTTGCGGATTTTGCT
677


 


miR-556-5p
4
GATGAGCTCATTGTAATATGAG
678


 


miR-581
4
TCTTGTGTTCTCTAGATCAGT
679


 


miR-584
4
TTATGGTTTGCCTGGGACTGAG
680


 


miR-590-3p
4
TAATTTTATGTATAAGCTAGT
681


 


miR-616
4
AGTCATTGGAGGGTTTGAGCAG
682


 


miR-629
4
TGGGTTTACGTTGGGAGAACT
683


 


miR-92a
4
TATTGCACTTGTCCCGGCCTGT
684


 


miR-941
4
CACCCGGCTGTGTGCACATGTGC
685


 


miR-942
4
TCTTCTCTGTTTTGGCCATGTG
686


 


miR-944
4
AAATTATTGTACATCGGATGAG
687


 


miR-1255b
5
CGGATGAGCAAAGAAAGTGGTT
688


 


miR-1257
5
AGTGAATGATGGGTTCTGACC
689


 


miR-1285
5
TCTGGGCAACAAAGTGAGACCT
690


 


miR-130b*
5
ACTCTTTCCCTGTTGCACTAC
691


 


miR-181c*
5
AACCATCGACCGTTGAGTGGAC
692


 


miR-186*
5
GCCCAAAGGTGAATTTTTTGGG
693


 


miR-18a
5
TAAGGTGCATCTAGTGCAGATAG
694


 


miR-18b
5
TAAGGTGCATCTAGTGCAGTTAG
695


 


miR-190b
5
TGATATGTTTGATATTGGGTT
696


 


miR-20b*
5
ACTGTAGTATGGGCACTTCCAG
697


 


miR-24-2*
5
TGCCTACTGAGCTGAAACACAG
698


 


miR-28-3p
5
CACTAGATTGTGAGCTCCTGGA
699


 


miR-30c-1*
5
CTGGGAGAGGGTTGTTTACTCC
700


 


miR-363
5
AATTGCACGGTATCCATCTGTA
701


 


miR-450a
5
TTTTGCGATGTGTTCCTAATAT
702


 


miR-548g
5
AAAACTGTAATTACTTTTGTAC
703


 


miR-548o
5
CCAAAACTGCAGTTACTTTTGC
704


 


miR-582-5p
5
TTACAGTTGTTCAACCAGTTACT
705


 


miR-664*
5
ACTGGCTAGGGAAAATGATTGGAT
706


 


miR-93
5
CAAAGTGCTGTTCGTGCAGGTAG
707









The microRNA precursor sequences for the microRNAs listed in Table 14 are shown in Table 15.







TABLE 15







Precursor sequences and chromosomal locations


(from human qenome NCBI36) of the microRNAs in Table 19



















SEQ ID


Gene name
Precusor Sequence 5′->3′
chrom
start
end
strand
NO
















hsa-mir-101-1
TGCCCTGGCTCAGTTATCACAGTGCTGATGCTGTCTATTCTAAAGGT
1
65296705
65296779
−1
708



ACAGTACTGTGATAACTGAAGGATGGCA







 


hsa-mir-101-2
ACTGTCCTTTTTCGGTTATCATGGTACCGATGCTGTATATCTGAAAG
9
4840297
4840375
1
709



GTACAGTACTGTGATAACTGAAGAATGGTGGT







 


hsa-mir-140
TGTGTCTCTCTCTGTGTCCTGCCAGTGGTTTTACCCTATGGTAGGTT
16
68524485
68524584
1
710



ACGTCATGCTGTTCTACCACAGGGTAGAACCACGGACAGGATACCGG








GGCACC







 


hsa-mir-142
GACAGTGCAGTCACCCATAAAGTAGAAAGCACTACTAACAGCACTGG
17
53763592
53763678
−1
711



AGGGTGTAGTGTTTCCTACTTTATGGATGAGTGTACTGTG







 


hsa-mir-143
GCGCAGCGCCCTGTCTCCCAGCCTGAGGTGCAGTGCTGCATCTCTGG
5
148788674
148788779
1
712



TCAGTTGGGAGTCTGAGATGAAGCACTGTAGCTCAGGAAGAGAGAAG








TTGTTCTGCAGC







 


hsa-mir-144
TGGGGCCCTGGCTGGGATATCATCATATACTGTAAGTTTGCGATGAG
17
24212677
24212762
−1
713



ACACTACAGTATAGATGATGTACTAGTCCGGGCACCCCC







 


hsa-mir-148a
GAGGCAAAGTTCTGAGACACTCCGACTCTGAGTATGATAGAAGTCAG
7
25956064
25956131
−1
714



TGCACTACAGAACTTTGTCTC







 


hsa-mir-1537
ACAGCTGTAATTAGTCAGTTTTCTGTCCTGTCCACACAGAAAACCGT
1
234082923
234082983
−1
715



CTAGTTACAGTTGT







 


hsa-mir-15a
CCTTGGAGTAAAGTAGCAGCACATAATGGTTTGTGGATTTTGAAAAG
13
49521256
49521338
−1
716



GTGCAGGCCATATTGTGCTGCCTCAAAAATACAAGG







 


hsa-mir-16-1
GTCAGCAGTGCCTTAGCAGCACGTAAATATTGGCGTTAAGATTCTAA
13
49521110
49521198
−1
717



AATTATCTCCAGTATTAACTGTGCTGCTGAAGTAAGGTTGAC







 


hsa-mir-16-2
GTTCCACTCTAGCAGCACGTAAATATTGGCGTAGTGAAATATATATT
3
161605227
161605307
1
718



AAACACCAATATTACTGTGCTGCTTTAGTGTGAC







 


hsa-mi-185
AGGGGGCGAGGGATTGGAGAGAAAGGCAGTTCCTGATGGTCCCCTCC
22
18400662
18400743
1
719



CCAGGGGCTGGCTTTCCTCTGGTCCTTCCCTCCCA







 


hsa-mir-2115
ACTGTCATCCCACTGCTTCCAGCTTCCATGACTCCTGATGGAGGAAT
3
48332854
48332953
−1
720



CACATGAATTCATCAGAATTCATGGAGGCTAGAAGCAGTATGAGGAT








CATTTA







 


hsa-mir-223
CCTGGCCTCCTGCAGTGCCACGCTCCGTGTATTTGACAAGCTGAGTT
X
65155437
65155546
1
721



GGACACTCCATGTGGTAGAGTGTCAGTTTGTCAAATACCCCAAGTGC








GGCACATGCTTACCAG







 


hsa-mir-27a
CTGAGGAGCAGGGCTTAGCTGCTTGTGAGCAGGGTCCACACCAAGTC
19
13808254
13808331
−1
722



GTGTTCACAGTGGCTAAGTTCCGCCCCCCAG







 


hsa-mir-30e
GGGCAGTCTTTGCTACTGTAAACATCCTTGACTGGAAGCTGTAAGGT
1
40992614
40992705
1
723



GTTCAGAGGAGCTTTCAGTCGGATGTTTACAGCGGCAGGCTGCCA







 


hsa-mir-338
TCTCCAACAATATCCTGGTGCTGAGTGATGACTCAGGCGACTCCAGC
17
76714278
76714344
−1
724



ATCAGTGATTTTGTTGAAGA







 


hsa-mir-374a
TACATCGGCCATTATAATACAACCTGATAAGTGTTATAGCACTTATC
X
73423846
73423917
−1
725



AGATTGTATTGTAATTGTCTGTGTA







 


hsa-mir-451
CTTGGGAATGGCAAGGAAACCGTTACCATTACTGAGTTTAGTAATGG
17
24212513
24212584
−1
726



TAATGGTTCTCTTGCTATACCCAGA







 


hsa-mir-486
GCATCCTGTACTGAGCTGCCCCGAGGCCCTTCATGCTGCCCAGCTCG
8
41637116
41637183
−1
727



GGGCAGCTCAGTACAGGATAC







 


hsa-mir-548e
TTATTAGGTTGGTACAAAAGCAATCGCGGTTTTTGCTATTACTTTTA
10
112738674
112738761
1
728



AAGGCAAAAACTGAGACTACTTTTGCACCAACCTGATAGAA







 


hsa-mir-548f-1
ATTAGGTTGGTGCAAAAGTAATCACAGTTTTTGACATTACTTTCAAA
10
56037640
56037723
−1
729



GACAAAAACTGTAATTACTTTTGGACCAACCTAATAG







 


hsa-mir-548f-2
TAATAACTATTAGGTTGGTGCGAACATAATTGCAGTTTTTATCATTA
2
212999232
212999329
−1
730



CTTTTAATGGCAAAAACTGTAATTACTTTTGCACCAACCTAATATTT








TAGT







 


 


hsa-mir-548f-3
ATTAGGTTGGTGCAAACCTAATTGCAATTTTTGCAGTTTTTTTAAGT
5
109877429
109877515
−1
731



AATTGCAAAAACTGTAATTACTTTTGCACCAACCTAATAC







 


hsa-mir-5481-4
GAGTTCTAACGTATTAGGTTGGTGCAAAAGTAATAGTGGTTTTTGCC
7
146706042
146706146
−1
732



ATTAAAAGTAATGACAAAAACTGTAATTACTTTTGGAACAATATTAA








TAGAATTTCAG







 


hsa-mir-548f-5
TATTAGGTTGCTGCAAAAGTAATCATGTTTTTTTCCATTGTAAGTAA
X
32569512
32569597
−1
733



TGGGAAAAACTGTAATTACTTTTGTACCAACCTAATAGC







 


hsa-mir-548j
GGGCAGCCAGTGAATAGTTAGCTGGTGCAAAAGTAATTGCGGTCTTT
22
25281178
25281289
−1
734



GGTATTACTTTCAGTGGCAAAAACTGCATTACTTTTGCACCAGCCTA








CTAGAACGCTGAGTTCAG







 


hsa-mir-548n
AGGTTGGTGCAAAAGTAATTGTGGATTTTGTCGTTAAAAATAGCAAA
7
34946897
34946971
−1
735



ACCCGCAATTACTTTTGCACCAACCTAA







 


hsa-mir-548q
ATATTAGGCTGGTGCAAAAGTAATGGCGGTTTTTGCCATTACTTTTC
10
12807259
12807358
−1
736



ATTTTTACCATTAAAAGTAATGGCAAAAAGCATGATTACTTTTTCAC








CAACCT







 


hsa-mir-576
TACAATCCAACGAGGATTCTAATTTCTCCACGTCTTTGGTAATAAGG
4
110629303
110629400
1
737



TTTGGCAAAGATGTGGAAAAATTGGAATCCTCATTCGATTGGTTATA








ACCA







 


hsa-mir-582
ATCTGTGCTCTTTGATTACAGTTGTTCAACCAGTTACTAATCTAACT
5
59035189
59035286
−1
738



AATTGTAACTGGTTGAACAACTGAACCCAAAGGGTGCAAAGTAGAAA








CATT







 


hsa-mir-617
CATCATAAGGAGCCTAGACTTCCCATTTGAAGGTGGCCATTTCCTAC
12
79750443
79750539
−1
739



CACCTTCAAATGGTAAGTCCAGGCTCCTTCTGATTCAATAAATGAGG








AGC







 


hsa-mir-618
CTCTTGTTCACAGCCAAACTCTACTTGTCCTTCTGAGTGTAATTACG
12
79853646
79853743
−1
740



TACATGCAGTAGCTCAGGAGACAAGCAGGTTTACCCTGTGGATGAGT








CTGA







 


hsa-mir-624
AATGCTGTTTCAAGGTAGTACCAGTACCTTGTGTTCAGTGGAACCAA
14
30553603
30553699
−1
741



GGTAAACACAAGGTATTGGTATTACCTTGAGATAGCATTACACCTAA








GTG







 


hsa-mir-625
AGGGTAGAGGGATGAGGGGGAAAGTTCTATAGTCCTGTAATTAGATC
14
65007573
65007657
1
742



TCAGGACTATAGAACTTTCCCCCTCATCCCTCTGCCCT







 


hsa-mir-627
TACTTATTACTGGTAGTGAGTCTCTAAGAAAAGAGGAGGTGGTTGTT
15
40279060
40279156
−1
743



TTCCTCCTCTTTTCTTTGAGACTCACTACCAATAATAAGAAATACTA








CTA







 


hsa-mir-628
ATAGCTGTTGTGTCACTTCCTCATGCTGACATATTTACTAGAGGGTA
15
53452430
53452524
−1
744



AAATTAATAACCTTCTAGTAAGAGTGGCAGTCGAAGGGAAGGGCTCA








T







 


hsa-mir-629
TCCCTTTCCCAGGGGAGGGGCTGGGTTTACGTTGGGAGAACTTTTAC
15
68158765
68158861
−1
745



GGTGAACCAGGAGGTTCTCCCAACGTAAGCCCAGCCCCTCCCCTCTG








CCT







 


hsa-mir-640
GTGACCCTGGGCAAGTTCCTGAAGATCAGACACATCAGATCCCTTAT
19
19406872
19406967
1
746



CTGTAAAATGGGCATGATCCAGGAACCTGCCTCTACGGTTGCCTTGG








GG







 


hsa-mir-1225
GTGGGTACGGCCCAGTGGGGGGGAGAGGGACACGCCCTGGGCTCTGC
16
2080197
2080286
−1
747



CCAGGGTGCAGCCGGACTGACTGAGCCCCTGTGCCGCCCCCAG







 


hsa-mir-1255a
ATTGGAAATCCTTTGAGTTGCTTCTCAAGGATGAGCAAAGAAAGTAG
4
102470482
102470594
−1
748



ATTTTTTAGATTCTAAAGAAACTATCTTCTTTGCTCATCCTTGAGAA








GCAACTCCTTATCCATTAA







 


hsa-mir-126
CGCTGGCGACGGGACATTATTACTTTTGGTACGCGCTGTGACACTTC
9
138684875
138684959
1
749



AAACTCGTACCGTGAGTAATAATGCGCCGTCCACGGCA







 


hsa-mir-148a
GAGGCAAAGTTCTGAGACACTCCGACTCTGAGTATGATAGAAGTCAG
7
25956064
25956131
−1
750



TGCACTACAGAACTTTGTCTC







 


hsa-mir-26b
CCGGGACCCAGTTCAAGTAATTCAGGATAGGTTGTGTGCTGTCCAGC
2
218975613
218975689
1
751



CTGTTCTCCATTACTTGGCTCGGGGACCGG







 


hsa-mir-30a
GCGACTGTAAACATCCTCGACTGGAAGCTGTGAAGCCACAGATGGGC
6
72169975
72170045
−1
752



TTTCAGTCGGATGTTTGCAGCTGC







 


hsa-mir-425
GAAAGCGCTTTGGAATGACACGATCACTCCCGTTGAGTGGGCACCCG
3
49032585
49032671
−1
753



AGAAGCCATCGGGAATGTCGTGTCCGCCCAGTGCTCTTTC







 


hsa-mir-450b
GCAGAATTATTTTTGCAATATGTTCCTGAATATGTAATATAAGTGTA
X
133501881
133501958
−1
754



TTGGGATCATTTTGCATCCATAGTTTTGTAT







 


hsa-mir-503
TGCCCTAGCAGCGGGAACAGTTCTGCAGTGAGCGATCGGTGCTCTGG
X
133508024
133508094
−1
755



GGTATTGTTTCCGCTGCCAGGGTA







 


hsa-mir-514-1
AACATGTTGTCTGTGGTACCCTACTCTGGAGAGTGACAATCATGTAT
X
146168457
146168554
−1
756



AATTAAATTTGATTGACACTTCTGTGAGTAGAGTAACGCATGACACG








TACG







 


hsa-mir-514-2
GTTGTCTGTGGTACCCTACTCTGGAGAGTGACAATCATGTATAACTA
X
146171153
146171240
−1
757



AATTTGATTGACACTTCTGTGAGTAGAGTAACGCATGACAC







 


hsa-mir-514-3
GTTGTCTGTGGTACCCTACTCTGGAGAGTGACAATCATGTATAACTA
X
146173851
146173938
−1
758



AATTTGATTGACACTTCTGTGAGTAGAGTAACGCATGACAC







 


hsa-mir-548b
CAGACTATATATTTAGGTTGGCGCAAAAGTAATTGTGGTTTTGGCCT
6
11943191
119432007
−1
759



TTATTTTCAATGGCAAGAACCTCAGTTGCTTTTGTGCCAACCTAATA








CTT







 


hsa-mir-548d-1
AAACAAGTTATATTAGGTTGGTGCAAAAGTAATTGTGGTTTTTGCCT
8
124429455
124429551
−1
760



GTAAAAGTAATGGCAAAAACCACAGTTTCTTTTGCACCAGACTAATA








AAG







 


hsa-mir-548d-2
GAGAGGGAAGATTTAGGTTGGTGCAAAAGTAATTGTGGTTTTTGCCA
17
62898067
62898163
−1
761



TTGAAAGTAATGGCAAAAACCACAGTTTCTTTTGCACCAACCTAATA








AAA







 


hsa-mir-580
ATAAAATTTCCAATTGGAACCTAATGATTCATCAGACTCAGATATTT
5
36183751
36183847
−1
762



AAGTTAACAGTATTTGAGAATGATGAATCATTAGGTTCCGGTCAGAA








ATT







 


hsa-mir-616
TTAGGTAATTCCTCCACTCAAAACCCTTCAGTGACTTCCATGACATG
12
56199213
56199309
−1
763



AAATAGGAAGTCATTGGAGGGTTTGAGCAGAGGAATGACCTGTTTTA








AAA







 


hsa-mir-1179
GGCTGGAAAGGAAGAAGCATTCTTTCATTGGTTGGTGTGTATTGCCT
15
86952342
86952432
1
764



TGTCAACCAATAAGAGGATGCCATTTATCCTTTTCTGACTAGCT







 


hsa-mir-1244
ATCTTATTCCGAGCATTCCAGTAACTTTTTTGTGTATGTACTTAGCT
12
12156153
12156237
1
765



GTACTATAAGTAGTTGGTTTGTATGAGATGGTTAAAAA







 


hsa-mir-1244
ATCTTATTCCGAGCATTCCAGTAACTTTTTTGTGTATGTACTTAGCT
12
9283330
9283414
−1
766



GTACTATAAGTAGTTGGTTTGTATGAGATGGTTAAAAA







 


hsa-mir-1244
ATCTTATTCCGAGCATTCCAGTAACTTTTTTGTGTATGTACTTAGCT
2
232286268
232286352
1
767



GTACTATAAGTAGTTGGTTTGTATGAGATGGTTAAAAA







 


hsa-mir-1244
ATCTTATTCCGAGCATTCCAGTAACTTTTTTGTGTATGTACTTAGCT
5
118338180
118338264
1
768



GTACTATAAGTAGTTGGTTTGTATGAGATGGTTAAAAA







 


hsa-mir-12
CGCTGGCGACGGGACATTATTACTTTTGGTACGCGCTGTGACACTTC
9
138684875
138684959
1
769



AAACTCGTACCGTGAGTAATAATGCGCCGTCCACGGCA







 


hsa-mir-1291
GGTAGAATTCCAGTGGCCCTGACTGAAGACCAGCAGTTGTACTGTGG
12
47334494
47334580
−1
770



CTGTTGGTTTCAAGCAGAGGCCTAAAGGACTGTCTTCCTG







 


hsa-miR-140
TGTGTCTCTCTCTGTGTCCTGCCAGTGGTTTTACCCTATGGTAGGTT
16
68524485
68524584
1
771



ACGTCATGCTGTTCTACCACAGGGTAGAACCACGGACAGGATACCGG








GGCACC







 


hsa-mir-145
CACCTTGTCCTCACGGTCCAGTTTTCCCAGGAATCCCTTAGATGCTA
5
148790402
148790489
1
772



AGATGGGGATTCCTGGAAATACTGTTCTTGAGGTCATGGTT







 


hsa-mir-146a
CCGATGTGTATCCTCAGCTTTGAGAACTGAATTCCATGGGTTGTGTC
5
159844937
159845035
1
773



AGTGTCAGACCTCTGAAATTCAGTTCTTCAGCTGGGATATCTCTGTC








ATCGT







 


hsa-mir-146b
CCTGGCACTGAGAACTGAATTCCATAGGCTGTGAGCTCTAGCAATGC
10
104186259
104186331
1
774



CCTGTGGACTCAGTTCTGGTGCCCGG







 


hsa-mir-148b
CAAGCACGATTAGCATTTGAGGTGAAGTTCTGTTATACACTCAGGCT
12
53017267
53017365
1
775



GTGGCTCTCTGAAAGTCAGTGCATCACAGAACTTTGTCTCGAAAGCT








TTCTA







 


hsa-mir-150
CTCCCCATGGCCCTGTCTCCCAACCCTTGTACCAGTGCTGGGCTCAG
19
54695854
54695937
−1
776



ACCCTGGTACAGGCCTGGGGGACAGGGACCTGGGGAC







 


hsa-mir-17
GTCAGAATAATGTCAAAGTGCTTACAGTGCAGGTAGTGATATGTGCA
13
90800860
90800943
1
777



TCTACTGCAGTGAAGGCACTTGTAGCATTATGGTGAC







 


hsa-mir-181a-1
TGAGTTTTGAGGTTGCTTCAGTGAACATTCAACGCTGTCGGTGAGTT
1
197094796
197094905
−1
778



TGGAATTAAAATCAAAACCATCGACCGTTGATTGTACCCTATGGCTA








ACCATCATCTACTCCA







 


hsa-mir-191
CGGCTGGACAGCGGGCAACGGAATCCCAAAAGCAGCTGTTGTCTCCA
3
49033055
49033146
−1
779



GAGCATTCCAGCTGCGCTTGGATTTCGTCCCCTGCTCTCCTGCCT







 


hsa-mir-199b
CCAGAGGACACCTCCACTCCGTCTACCCAGTGTTTAGACTATCTGTT
9
130046821
130046930
−1
780



CAGGACTCCCAAATTGTACAGTAGTCTGCACATTGGTTAGGCTGGGC








TGGGTTAGACCCTCGG







 


hsa-mir-26a-1
GTGGCCTCGTTCAAGTAATCCAGGATAGGCTGTGCAGGTCCCAATGG
3
37985899
37985975
1
781



GCCTATTCTTGGTTACTTGCACGGGGACGC







 


hsa-mir-29b-2
CTTCTGGAAGCTGGTTTCACATGGTGGCTTAGATTTTTCCATCTTTG
1
206042411
206042491
−1
782



TATCTAGCACCATTTGAAATCAGTGTTTTAGGAG







 


hsa-mir-340
TTGTACCTGGTGTGATTATAAAGCAATGAGACTGATTGTCATATGTC
5
179374909
179375003
−1
783



GTTTGTGGGATCCGTCTCAGTTACTTTATAGCCATACCTGGTATCTT








A







 


hsa-mir-361
GGAGCTTATCAGAATCTCCAGGGGTACTTTATAATTTCAAAAAGTCC
X
85045297
85045368
−1
784



CCCAGGTGTGATTCTGATTTGCTTC







 


hsa-mir-422a
GAGAGAAGCACTGGACTTAGGGTCAGAAGGCCTGAGTCTCTCTGCTG
15
61950182
61950271
−1
785



CAGATGGGCTCTCTGTCCCTGAGCCAAGCTTTGTCCTCCCTGG







 


hsa-mir-425
GAAAGCGCTTTGGAATGACACGATCACTCCCGTTGAGTGGGCACCCG
3
49032585
49032671
−1
786



AGAAGCCATCGGGAATGTCGTGTCCGCCCAGTGCTCTTTC







 


hsa-mir-454
TCTGTTTATCACCAGATCCTAGAACCCTATCAATATTGTCTCTGCTG
17
54569901
54570015
−1
787



TGTAAATAGTTCTGAGTAGTGCAATATTGCTTATAGGGTTTTGGTGT








TTGGAAAGAACAATGGGCAGG







 


hsa-mir-502
TGCTCCCCCTCTCTAATCCTTGCTATCTGGGTGCTAGTGCTGGCTCA
X
49665946
49666031
1
788



ATGCAATGCACCTGGGCAAGGATTCAGAGAGGGGGAGCT







 


hsa-mir-542
CAGATCTCAGACATCTCGGGGATCATCATGTCACGAGATACCAGTGT
X
133503037
133503133
−1
789



GCACTTGTGACAGATTGATAACTGAAAGGTCTGGGAGCCACTCATCT








TCA







 


hsa-mir-550-1
TGATGCTTTGCTGGCTGGTGCAGTGCCTGAGGGAGTAAGAGCCCTGT
7
30295935
30296031
1
790



TGTTGTAAGATAGTGTCTTACTCCCTCAGGCACATCTCCAACAAGTC








TCT







 


hsa-mir-550-2
TGATGCTTTGCTGGCTGGTGCAGTGCCTGAGGGAGTAAGAGCCCTGT
7
32739118
32739214
1
791



TGTTGTCAGATAGTGTCTTACTCCCTCAGGCACATCTCCAGCGAGTC








TCT







 


hsa-mir-551a
GGGGACTGCCGGGTGACCCTGGAAATCCAGAGTGGGTGGGGCCAGTC
1
3467119
3467214
−1
792



TGACCGTTTCTAGGCGACCCACTCTTGGTTTCCAGGGTTGCCCTGGA








AA







 


hsa-mir-573
TTTAGCGGTTTCTCCCTGAAGTGATGTGTAACTGATCAGGATCTACT
4
24130913
24131011
−1
793



CATGTCGTCTTTGGTAAAGTTATGTCGCTTGTCAGGGTGAGGAGAGT








TTTTG







 


hsa-mir-598
GCTTGATGATGCTGCTGATGCTGGCGGTGATCCCGATGGTGTGAGCT
8
10930126
10930222
−1
794



GGAAATGGGGTGCTACGTCATCGTTGTCATCGTCATCATCATCATCC








GAG







 


hsa-mir-643
ACCAAGTGATATTCATTGTCTACCTGAGCTAGAATACAAGTAGTTGG
19
57476862
57476958
1
795



CGTCTTCAGAGACACTTGTATGCTAGCTCAGGTAGATATTGAATGAA








AAA







 


hsa-mir-671
GCAGGTGAACTGGCAGGCCAGGAAGAGGAGGAAGCCCTGGAGGGGCT
7
150566440
150566557
1
796



GGAGGTGATGGATGTTTTCCTCCGGTTCTCAGGGCTCCACCTCTTTC








GGGCCGTAGAGCCAGGGCTGGTGC







 


hsa-let-7f-1
TCAGAGTGAGGTAGTAGATTGTATAGTTGTGGGGTAGTGATTTTACC
9
95978450
95978536
1
797



CTGTTCAGGAGATAACTATACAATCTATTGCCTTCCCTGA







 


hsa-let-7f-2
TGTGGGATGAGGTAGTAGATTGTATAGTTTTAGGGTCATACCCCATC
X
53600878
53600960
−1
798



TTGGAGATAACTATACAGTCTACTGTCTTTCCCACG







 


hsa-let-7g
AGGCTGAGGTAGTAGTTTGTACAGTTTGAGGGTCTATGATACCACCC
3
52277334
52277417
−1
799



GGTACAGGAGATAACTGTACAGGCCACTGCCTTGCCA







 


hsa-mir-103-1
TACTGCCCTCGGCTTCTTTACAGTGCTGCCTTGTTGCATATGGATCA
5
167920479
167920556
−1
800



AGCAGCATTGTACAGGGCTATGAAGGCATTG







 


hsa-mir-103-2
TTGTGCTTTCAGCTTCTTTACAGTGCTGCCTTGTAGCATTCAGGTCA
20
3846141
3846218
1
801



AGCAGCATTGTACAGGGCTATGAAAGAACCA







 


hsa-mir-107
CTCTCTGCTTTCAGCTTCTTTACAGTGTTGCCTTGTGGCATGGAGTT
10
91342484
91342564
−1
802



CAAGCAGCATTGTACAGGGCTATCAAAGCACAGA







 


hsa-mir-1197
ACTTCCTGGTATTTGAAGATGCGGTTGACCATGGTGTGTACGCTTTA
14
100561654
100561741
1
803



TTTGTGACGTAGGACACATGGTCTACTTCTTCTCAATATCA







 


hsa-mir-1250
CTGTCCCGCTGGCCTGGCAGGTGACGGTGCTGGATGTGGCCTTTTTG
17
76721591
76721703
−1
804



CCTTTTCTAAAGGCCACATTTTCCAGCCCATTCAACCTTCCAGAGCC








CTCTGAAGTGGCCACAGGC







 


hsa-mir-1277
ACCTCCCAAATATATATATATATGTACGTATGTGTATATAAATGTAT
X
117404385
117404462
1
805



ACGTAGATATATATGTATTTTTGGTGGGTTT







 


hsa-mir-1278
ATTTGCTCATAGATGATATGCATAGTACTCCCAGAACTCATTAAGTT
−1
1913722576
191372336
1
806



GGTAGTACTGTGCATATCATCTATGAGCGAATAG







 


hsa-mir-1299
CCTCATGGCAGTGTTCTGGAATCCTACGTGAGGGACAATCATTCAGA
9
68292059
68292141
−1
807



CCCACGTAGCAGTGTTCTGGAATTCTGTGTGAGGGA







 


hsa-mir-143
GCGCAGCGCCCTGTCTCCCAGCCTGAGGTGCAGTGCTGCATCTCTGG
5
148788674
148788779
1
808



TCAGTTGGGAGTCTGAGATGAAGCACTGTAGCTCAGGAAGAGAGAAG








TTGTTCTGCAGC







 


hsa-mir-150
CTCCCCATGGCCCTGTCTCCCAACCCTTGTACCAGTGCTGGGCTCAG
19
54695854
54695937
−1
809



ACCCTGGTACAGGCCTGGGGGACAGGGACCTGGGGAC







 


hsa-mir-155
CTGTTAATGCTAATCGTGATAGGGGTTTTTGCCTCCAACTGACTCCT
21
25868163
25868227
1
810



ACATATTAGCATTAACAG







 


hsa-mir-181a-1
TGAGTTTTGAGGTTGCTTCAGTGAACATTCAACGCTGTCGGTGAGTT
1
197094796
197094905
−1
811



TGGAATTAAAATCAAAACCATCGACCGTTGATTGTACCCTATGGCTA








ACCATCATCTACTCCA







 


hsa-mir-181a-2
AGAAGGGCTATCAGGCCAGCCTTCAGAGGACTCCAAGGAACATTCAA
9
126494542
126494651
1
812



CGCTGTCGGTGAGTTTGGGATTTGAAAAAACCACTGACCGTTGACTG








TACCTTGGGGTCCTTA







 


hsa-mir-181b-1
CCTGTGCAGAGATTATTTTTTAAAAGGTCACAATCAACATTCATTGC
1
197094625
197094734
−1
813



TGTCGGTGGGTTGAACTGTGTGGACAAGCTCACTGAACAATGAATGC








AACTGTGGCCCCGCTT







 


hsa-mir-181b-2
CTGATGGCTGCACTCAACATTCATTGCTGTCGGTGGGTTTGAGTCTG
9
126495810
126495898
1
814



AATCAACTCACTGATCAATGAATGCAAACTGCGGACCAAACA







 


hsa-mir-186
TGCTTGTAACTTTCCAAAGAATTCTCCTTTTGGGCTTTCTGGTTTTA
1
71305902
71305987
−1
815



TTTTAAGCCCAAAGGTGAATTTTTTGGGAAGTTTGAGCT







 


hsa-mir-196b
ACTGGTCGGTGATTTAGGTAGTTTCCTGTTGTTGGGATCCACCTTTC
7
27175624
27175707
−1
816



TCTCGACAGCACGACACTGCCTTCATTACTTCAGTTG







 


hsa-mir-1976
GCAGCAAGGAAGGCAGGGGTCCTAAGGTGTGTCCTCCTGCCCTCCTT
1
26753620
26753671
1
817



GCTGT







 


hsa-mir-21
TGTCGGGTAGCTTATCAGACTGATGTTGACTGTTGAATCTCATGGCA
17
55273409
55273480
1
818



ACACCAGTCGATGGGCTGTCTGACA







 


hsa-mir-219-1
CCGCCCCGGGCCGCGGCTCCTGATTGTCCAAACGCAATTCTCGAGTC
6
33283590
33283699
1
819



TATGGCTCCGGCCGAGAGTTGAGTCTGGACGTCCCGAGCCGCCGCCC








CCAAACCTCGAGCGGG







 


hsa-mir-221
TGAACATCCAGGTCTGGGGCATGAACCTGGCATACAATGTAGATTTC
X
45490529
45490638
−1
820



TGTGTTCGTTAGGCAACAGCTACATTGTCTGCTGGGTTTCAGGCTAC








CTGGAAACATGTTCTC







 


hsa-mir-378
AGGGCTCCTGACTCCAGGTCCTGTGTGTTACCTAGAAATAGCACTGG
5
149092581
149092646
1
821



ACTTGGAGTCAGAAGGCCT







 


hsa-mir-423
ATAAAGGAAGTTAGGCTGAGGGGCAGAGAGCGAGACTTTTCTATTTT
17
25468223
25468316
1
822



CCAAAAGCTCGGTCTGAGGCCCCTCAGTCTTGCTTCCTAACCCGCGC







 


hsa-mir-500
GCTCCCCCTCTCTAATCCTTGCTACCTGGGTGAGAGTGCTGTCTGAA
X
49659779
49659862
1
823



TGCAATGCACCTGGGCAAGGATTCTGAGAGCGAGAGC







 


hsa-mir-502
TGCTCCCCCTCTCTAATCCTTGCTATCTGGGTGCTAGTGCTGGCTCA
X
49665946
49666031
1
824



ATGCAATGCACCTGGGCAAGGATTCAGAGAGGGGGAGCT







 


hsa-mir-532
CGACTTGCTTTCTCTCCTCCATGCCTTGAGTGTAGGACCGTTGGCAT
X
49654494
49654584
1
825



CTTAATTACCCTCCCACACCCAAGGCTTGCAAAAAAGCGAGCCT







 


hsa-mir-548k
CTTTTCTCAAGTATTGCTGTTAGGTTGGTGCAAAAGTACTTGCGGAT
11
69807709
69807824
1
826



TTTGCTTTACTTTTAATGGCAAAAACCGCAATTATTTTTGCTTCAAC








CTAATATGATGCAAAATTGGCT







 


hsa-mir-556
GATAGTAATAAGAAAGATGAGCTCATTGTAATATGAGCTTCATTTAT
1
160578960
160579054
1
827



ACATTTCATATTACCATTAGCTCATCTTTTTTATTACTACCTTCAAC








A







 


hsa-mir-581
GTTATGTGAAGGTATTCTTGTGTTCTCTAGATCAGTGCTTTTAGAAA
5
53283091
53283186
−1
828



ATTTGTGTGATCTAAAGAACACAAAGAATACCTACACAGAACCACCT








GC







 


hsa-mir-584
TAGGGTGACCAGCCATTATGGTTTGCCTGGGACTGAGGAATTTGCTG
5
148422069
148422165
−1
829



GGATATGTCAGTTCCAGGCCAACCAGGCTGGTTGGTCTCCCTGAAGC








AAC







 


hsa-mir-590
TAGCCAGTCAGAAATGAGCTTATTCATAAAAGTGCAGTATGGTGAAG
7
73243464
73243560
1
830



TCAATCTGTAATTTTATGTATAAGCTAGTCTCTGATTGAAACATGCA








GCA







 


hsa-mir-616
TTAGGTAATTCCTCCACTCAAAACCCTTCAGTGACTTCCATGACATG
12
56199213
56199309
−1
831



AAATAGGAAGTCATTGGAGGGTTTGAGCAGAGGAATGACCTGTTTTA








AAA







 


hsa-mir-629
TCCCTTTCCCAGGGGAGGGGCTGGGTTTACGTTGGGAGAACTTTTAC
15
68158765
68158861
−1
832



GGTGAACCAGGAGGTTCTCCCAACGTAAGCCCAGCCCCTCCCCTCTG








CCT







 


hsa-mir-92a-1
CTTTCTACACAGGTTGGGATCGGTTGCAATGCTGTGTTTCTGTATGG
13
90801569
90801646
1
833



TATTGCACTTGTCCCGGCCTGTTGAGTTTGG







 


hsa-mir-92a-2
TCATCCCTGGGTGGGGATTTGTTGCATTACTTGTGTTCTATATAAAG
X
133131234
133131308
−1
834



TATTGCACTTGTCCCGGCCTGTGGAAGA







 


hsa-mir-941-1
CCCGGCTGTGTGGACATGTGCCCAGGGCCCGGGACAGCGCCACGGAA
20
62021238
62021326
1
835



GAGGACGCACCCGGCTGTGTGCACATGTGCCCAGGGCCCGGG







 


hsa-mir-941-2
CCCGGCTGTGTGCACATGTGCCCAGGGCCCGGGACAGCGCCACGGAA
20
62021545
62021633
1
836



GAGGACGCACCCGGCTGTGTGCACATGTGCCCAGGGCCCGGG







 


hsa-mir-941-3
CCCGGCTGTGTGCACATGTGCCCAGGGCCCGGGACAGCGCCACGGAA
20
62021657
62021745
1
837



GAGGACGCACCCGGCTGTGTGCACATGTGCCCAGGGCCCGGG







 


hsa-mir-942
ATTAGGAGAGTATCTTCTCTGTTTTGGCCATGTGTGTACTCACAGCC
1
117438788
117438873
1
838



CCTCACACATGGCCGAAACAGAGAAGTTACTTTCCTAAT







 


hsa-mir-944
GTTCCAGACACATCTCATCTGATATACAATATTTTCTTAAATTGTAT
3
191030405
191030492
1
839



AAAGAGAAATTATTGTACATCGGATGAGCTGTGTCTGGGAT







 


hsa-mir-1255b-1
TACGGATGAGCAAAGAAAGTGGTTTCTTAAAATGGAATCTACTCTTT
4
36104383
36104445
−1
840



GTGAAGATGCTGTGAA







 


hsa-mir-1255b-2
TCTTACGGATGAGCAAAGAAAGTGGTTTGCGCCTCAAGAAACCACTT
1
166234522
166234588
1
841



TCTTTGCTCATCCATAAGGA







 


hsa-mir-1257
GCCCTGGGCTTGTGCTTGGGGAGTGAATGATGGGTTCTGACCCCCAT
20
59961997
59962113
−1
842



GCACCCCTGTGGGCCCCTGGCATCACTGGCCCCATCCTTCACCCCTG








CCAACCACGCTTGCCCTGTGCCT







 


hsa-mir-1285-1
TGTAGAGATAGGATCTCACTTTGTTGCCCAGGCTGGTCTCAAACTCC
7
91671265
91671348
−1
843



TGGTCTGGGCAACAAAGTGAGACCTTATCTCTACAAG







 


hsa-mir-1285-2
TTTGGGAGGCCGAGGCTGGTGCATCACTTGAGCCCAGCAATTTGAGA
2
70333554
70333641
−1
844



CCAATCTGGGCAACAAAGTGAGACCTCCGTCTCTACAAAGA







 


hsa-mir-130b
GGCCTGCCCGACACTCTTTCCCTGTTGCACTACTATAGGCCGCTGGG
22
20337593
20337674
1
845



AAGCAGTGCAATGATGAAAGGGCATCGGTCAGGTC







 


hsa-mir-181c
CGGAAAATTTGCCAAGGGTTTGGGGGAACATTCAACCTGTCGGTGAG
19
13846513
13846622
1
846



TTTGGGCAGCTCAGGCAAACCATCGACCGTTGAGTGGACCCTGAGGC








CTGGAATTGCCATCCT







 


hsa-mir-186
TGCTTGTAACTTTCCAAAGAATTCTCCTTTTGGGCTTTCTGGTTTTA
1
71305902
71305987
−1
847



TTTTAAGCCCAAAGGTGAATTTTTTGGGAAGTTTGAGCT







 


hsa-mir-18a
TGTTCTAAGGTGCATCTAGTGCAGATAGTGAAGTAGATTAGCATCTA
13
90801006
90801076
1
848



CTGCCCTAAGTGCTCCTTCTGGCA







 


hsa-mir-18b
TGTGTTAAGGTGCATCTAGTGCAGTTAGTGAAGCAGCTTAGAATCTA
X
133131737
133131807
−1
849



CTGCCCTAAATGCCCCTTCTGGCA







 


hsa-mir-190b
TGCTTCTGTGTGATATGTTTGATATTGGGTTGTTTAATTAGGAACCA
1
152432765
152432843
−1
850



ACTAAATGTCAAACATATTCTTACAGCAGCAG







 


hsa-mir-20b
AGTACCAAAGTGCTCATAGTGCAGGTAGTTTTGGCATGACTCTACTG
X
133131505
133131573
−1
851



TAGTATGGGCACTTCCAGTACT







 


hsa-mir-24-2
CTCTGCCTCCCGTGCCTACTGAGCTGAAACACAGTTGGTTTGTGTAC
19
13808101
13808173
−1
852



ACTGGCTCAGTTCAGCAGGAACAGGG







 


hsa-mir-28
GGTCCTTGCCCTCAAGGAGCTCACAGTCTATTGAGTTACCTTTCTGA
3
189889263
189889348
1
853



CTTTCCCACTAGATTGTGAGCTCCTGGAGGGCAGGCACT







 


hsa-mir-30c-1
ACCATGCTGTAGTGTGTGTAAACATCCTACACTCTCAGCTGTGAGCT
1
40995543
40995631
1
854



CAAGGTGGCTGGGAGAGGGTTGTTTACTCCTTCTGCCATGGA







 


hsa-mir-363
TGTTGTCGGGTGGATCACGATGCAATTTTGATGAGTATCATAGGAGA
X
133131074
133131148
−1
855



AAAATTGCACGGTATCCATCTGTAAACC







 


hsa-mir-450a-1
AAACGATACTAAACTGTTTTTGCGATGTGTTCCTAATATGCACTATA
X
133502037
133502127
−1
856



AATATATTGGGAACATTTTGCATGTATAGTTTTGTATCAATATA







 


hsa-mir-450a-2
CCAAAGAAAGATGCTAAACTATTTTTGCGATGTGTTCCTAATATGTA
X
133502204
133502303
−1
857



ATATAAATGTATTGGGGACATTTTGCATTCATAGTTTTGTATCAATA








ATATGG







 


hsa-mir-548g
AGTTATTAGATTAGTGCAAAAGTAATTGCAGTTTTTGCATTACGTTC
4
148485231
148485319
−1
858



TATGGCAAAACTGTAATTACTTTTGTACCAACATAATACTTC







 


hsa-mir-548o
TGGTGAAAATGTGTTGATTGTAATGGTTCCTATTCTGATCAATAAAC
7
101833194
101833307
−1
859



ATGGTTTGAGCCTAGTTACAATGATCTAAAATTCACGGTCCAAAACT








GCAGTTACTTTTGCACCAAC







 


hsa-mir-582
ATCTGTGCTCTTTGATTACAGTTGTTCAACCAGTTACTAATCTAACT
5
59035189
59035286
−1
860



AATTGTAACTGGTTGAACAACTGAACCCAAAGGGTGCAAAGTAGAAA








CATT







 


hsa-mir-664
GAACATTGAAACTGGCTAGGGAAAATGATTGGATAGAAACTATTATT
1
218440503
218440584
−1
861



CTATTCATTTATCCCCAGCCTACAAAATGAAAAAA







 


hsa-mir-93
CTGGGGGCTCCAAAGTGCTGTTCGTGCAGGTAGTGTGATTACCCAAC
7
99529327
99529406
−1
862



CTACTGCTGAGCTAGCACTTCCCGAGCCCCCGG









Finally, a similar analysis identified novel star forms of microRNAs in miRBase (human version 14.0) that are present at increased levels in the sepsis smRNASeq dataset. While the precursor and mature microRNA are in miRBase, the novel star forms identified in this analysis are not. Again, the microRNAs were ranked from 1 to 5, depending on whether the sepsis patient smRNASeq dataset contained the highest number of counts of the particular microRNA, the second highest, etc. The results are shown in Table 16.







TABLE 16







Novel star-form microRNAs upregulated


in a sepsis smRNASeq dataset












microRNA
SEQ ID


microRNA name
RANK
Sequence 5′->3′
NO





mir-1537-STAR-5P
1
CTGTAATTAGTCAGTTTTCTG
863




T



 


mir-451-STAR-3P
1
TAATGGTAATGGTTCTCTTG
864


 


mir-580-STAR-5P
1
TAATGATTCATCAGACTCAGA
865


 


mir-606-STAR-5P
1
TTAGTAGTTTTACTATGATGA
866




GG



 


mir-643-STAR-5P
1
ACCTGAGCTAGAATACAAGTA
867




GT



 


mir-659-STAR-5P
1
AGGACCTTCCCTGAACCAAGG
868




A



 


mir-1284-STAR-3P
2
GAAAGCCCATGTTTGTATTGG
869




A



 


mir-215-STAR-3P
2
TCTGTCATTTCTTTAGGCCAA
870




TA



 


mir-345-STAR-3P
2
CCCTGAACGAGGGGTCTGGAG
871


 


mir-548j-STAR-3P
2
CAAAAACTGCATTACTTTTGC
872




A



mir-642-STAR-3P
2
AGACACATTTGGAGAGGGAAC
873


 


mir-1277-STAR-5P
3
TATATATATATATGTACGTAT
874




G



 


mir-548a-1-STAR-5P
3
GTTGGTGCAAAAGTAATTG
875


 


mir-548f-1-STAR-5P
3
GTTGGTGCAAAAGTAAT
876


 


mir-548h-1-STAR-3P
3
AAACTGGAATTACTTTTG
877


 


mir-597-STAR-3P
3
AGTGGTTCTCTTGTGGCTCAA
878




G



 


mir-939-STAR-3P
3
CTGGGCCTCTGCTCCCCAG
879


 


mir-1285-1-STAR-5P
4
TCTCACTTTGTTGCCCAG
880


 


mir-1975-STAR-5P
4
TGTGGGTTATTGTTAAGT
881


 


mir-2277-STAR-5P
4
AGCGCGGGCTGAGCGCTGCCA
882




GTC



 


mir-376a-2-STAR-5P
4
GTAGATTTTCCTTCTATGGTT
883


 


mir-421-STAR-5P
4
CTCATTAAATGTTTGTTGAAT
884




GA



 


mir-548e-STAR-5P
4
CAAAAGCAATCGCGGTTTTTG
885




C



 


mir-548f-4-STAR-5P
4
GTTGGTGCAAAAGTAAT
886


 


mir-941-1-STAR-5P
4
ACATGTGCCCAGGGCCCGGGA
887




CAGCG



 


mir-941-2-STAR-5P
4
ACATGTGCCCAGGGCCCGGGA
888




CAGCG



 


mir-941-3-STAR-5P
4
ACATGTGCCCAGGGCCCGGGA
889




CAGCG



 


mir-1234-STAR-5P
5
GGGGGGGGGGGGGGGGGG
890


 


mir-1273-STAR-5P
5
AGGAGAATTGCTTGAACCC
891


 


mir-1307-STAR-5P
5
TCGACCGGACCTCGACCGGCT
892


 


mir-153-2-STAR-5P
5
GTCATTTTTGTGATGTTGCAG
893




CT



 


mir-181b-1-STAR-3P
5
CTCACTGAACAATGAATGCA
894


 


mir-548h-3-STAR-3P
5
CAAAAACTGCAATTACTTTTG
895


 


mir-548h-4-STAR-3P
5
CAAAAACCGCAATTACTTTTG
896


 


mir-548l-STAR-3P
5
CAAAAACTGCAGTTACTT
897









The microRNA precursor sequences for the microRNAs listed in Table 16 are shown in Table 17.







TABLE 17







Precursor sequences and chromosomal locations


(from human qenome NCBI36) of the microRNAs in Table 16



















SEQ ID


Gene name
Precusor Sequence 5′->3′
chrom
start
end
strand
NO
















hsa-mir-1537
ACAGCTGTAATTAGTCAGTTTTCTGTCCTGTCCACACAGAAAACCGTC
1
234082923
234082983
−1
898



TAGTTACAGTTGT







 


hsa-mir-451
CTTGGGAATGGCAAGGAAACCGTTACCATTACTGAGTTTAGTAATGGT
17
24212513
24212584
−1
899



AATGGTTCTCTTGCTATACCCAGA







 


hsa-mir-580
ATAAAATTTCCAATTGGAACCTAATGATTCATCAGACTCAGATATTTA
5
36183751
36183847
−1
900



AGTTAACAGTATTTGAGAATGATGAATCATTAGGTTCCGGTCAGAAAT








T







 


hsa-mir-606
TGTATCCTTGGTTTTTAGTAGTTTTACTATGATGAGGTGTGCCATCCA
10
76982222
76982317
1
901



CCCCATCATAGTAAACTACTGAAAATCAAAGATACAAGTGCCTGACCA







 


hsa-mir-643
ACCAAGTGATATTCATTGTCTACCTGAGCTAGAATACAAGTAGTTGGC
19
57476862
57476958
1
902



GTCTTCAGAGACACTTGTATGCTAGCTCAGGTAGATATTGAATGAAAA








A







 


hsa-mir-659
TACCGACCCTCGATTTGGTTCAGGACCTTCCCTGAACCAAGGAAGAGT
22
36573631
36573727
−1
903



CACAGTCTCTTCCTTGGTTCAGGGAGGGTCCCCAACAATGTCCTCATG








G







 


hsa-mir-1284
ATTTTGATATATAAGCCAGTTTAATGTTTTCTATACAGACCCTGGCTT
3
71673811
71673930
−1
904



TTCTTAAATTTTATATATTGGAAAGCCCATGTTTGTATTGGAAACTGC








TGGTTTCTTTCATACTGAAAATCT







 


hsa-mir-215
ATCATTCAGAAATGGTATACAGGAAAATGACCTATGAATTGACAGACA
1
218357818
218357927
−1
905



ATATAGCTGAGTTTGTCTGTCATTTCTTTAGGCCAATATTCTGTATGA








CTGTGCTACTTCAA







 


hsa-mir-345
ACCCAAACCCTAGGTCTGCTGACTCCTAGTCCAGGGCTCGTGATGGCT
14
99843949
99844046
1
906



GGTGGGCCCTGAACGAGGGGTCTGGAGGCCTGGGTTTGAATATCGACA








GC







 


hsa-mir-548j
GGGCAGCCAGTGAATAGTTAGCTGGTGCAAAAGTAATTGCGGTCTTTG
22
25281178
25281289
−1
907



GTATTACTTTCAGTGGCAAAAACTGCATTACTTTTGCACCAGCCTACT








AGAACGCTGAGTTCAG







 


hsa-mir-642
ATCTGAGTTGGGAGGGTCCCTCTCCAAATGTGTCTTGGGGTGGGGGAT
19
50870026
50870122
1
908



CAAGACACATTTGGAGAGGGAACCTCCCAACTCGGCCTCTGCCATCAT








T







 


hsa-mir-1277
ACCTCCCAAATATATATATATATGTACGTATGTGTATATAAATGTATA
X
117404385
117404462
1
909



CGTAGATATATATGTATTTTTGGTGGGTTT







 


hsa-mir-548a-1
TGCAGGGAGGTATTAAGTTGGTGCAAAAGTAATTGTGATTTTTGCCAT
6
18679994
18680090
1
910



TAAAAGTAACGACAAAACTGGCAATTACTTTTGCACCAAACCTGGTAT








T







 


hsa-mir-548f-1
ATTAGGTTGGTGCAAAAGTAATCACAGTTTTTGACATTACTTTCAAAG
10
56037640
56037723
−1
911



ACAAAAACTGTAATTACTTTTGGACCAACCTAATAG







 


hsa-mir-548h-1
TCTGTCCATTAGGTGGGTGCAAAAGTAATCGCGGTTTTTGTCATTACT
14
63631495
63631596
−1
912



TTTAATGGTAAAAACTGGAATTACTTTTGCACTGACCTAATATTAAGC








CAGATA







 


hsa-mir-597
TACTTACTCTACGTGTGTGTCACTCGATGACCACTGTGAAGACAGTAA
8
9636592
9636688
1
913



AATGTACAGTGGTTCTCTTGTGGCTCAAGCGTAATGTAGAGTACTGGT








C







 


hsa-mir-939
TGTGGGCAGGGCCCTGGGGAGCTGAGGCTCTGGGGGTGGCCGGGGCTG
8
145590172
145590253
−1
914



ACCCTGGGCCTCTGCTCCCCAGTGTCTGACCGCG







 


hsa-mir-1285-1
TGTAGAGATAGGATCTCACTTTGTTGCCCAGGCTGGTCTCAAACTCCT
7
91671265
91671348
−1
915



GGTCTGGGCAACAAAGTGAGACCTTATCTCTACAAG







 


hsa-mir-1975
AGTTGGTCCGAGTGTTGTGGGTTATTGTTAAGTTGATTTAACATTGTC
7
148269513
148269587
1
916



TCCCCCCACAACCGCGCTTGACTAGCT







 


hsa-mir-2277
GTGCTTCCTGCGGGCTGAGCGCGGGCTGAGCGCTGCCAGTCAGCGCTC
5
92982158
92982250
−1
917



ACATTAAGGCTGACAGCGCCCTGCCTGGCTCGGCCGGCGAAGCTC







 


hsa-mir-376a-2
GGTATTTAAAAGGTAGATTTTCCTTCTATGGTTACGTGTTTGATGGTT
14
100576159
100576238
1
913



AATCATAGAGGAAAATCCACGTTTTCAGTATC







 


hsa-mir-421
GCACATTGTAGGCCTCATTAAATGTTTGTTGAATGAAAAAATGAATCA
X
73354937
73355021
−1
919



TCAACAGACATTAATTGGGCGCCTGCTCTGTGATCTC







 


hsa-mir-548e
TTATTAGGTTGGTACAAAAGCAATCGCGGTTTTTGCTATTACTTTTAA
10
112738674
112738761
1
920



AGGCAAAAACTGAGACTACTTTTGCACCAACCTGATAGAA







 


hsa-mir-548f-4
GAGTTCTAACGTATTAGGTTGGTGCAAAAGTAATAGTGGTTTTTGCCA
7
146706042
146706146
−1
921



TTAAAAGTAATGACAAAAACTGTAATTACTTTTGGAACAATATTAATA








GAATTTCAG







 


hsa-mir-941-1
CCCGGCTGTGTGGACATGTGCCCAGGGCCCGGGACAGCGCCACGGAAG
20
62021238
62021326
1
922



AGGACGCACCCGGCTGTGTGCACATGTGCCCAGGGCCCGGG







 


hsa-mir-941-2
CCCGGCTGTGTGCACATGTGCCCAGGGCCCGGGACAGCGCCACGGAAG
20
62021545
62021633
1
923



AGGACGCACCCGGCTGTGTGCACATGTGCCCAGGGCCCGGG







 


hsa-mir-941-3
CCCGGCTGTGTGCACATGTGCCCAGGGCCCGGGACAGCGCCACGGAAG
20
62021657
62021745
1
924



AGGACGCACCCGGCTGTGTGCACATGTGCCCAGGGCCCGGG







 


hsa-mir-1234
GTGAGTGTGGGGTGGCTGGGGGGGGGGGGGGGGGGCCGGGGACGGCTT
8
145596284
145596367
−1
925



GGGCCTGCCTAGTCGGCCTGACCACCCACCCCACAG







 


hsa-mir-1273
TGAGGCAGGAGAATTGCTTGAACCCGGGTGGTGGAGGTTGCAGTGAGC
8
101105386
101105488
−1
926



CAAGATTGCGCCACTGCACTCCAGCCTGGGCGACAAAGCAAGACTCTT








TCTTGGA







 


hsa-mir-1307
CATCAAGACCCAGCTGAGTCACTGTCACTGCCTACCAATCTCGACCGG
10
105144000
105144148
−1
927



ACCTCGACCGGCTCGTCTGTGTTGCCAATCGACTCGGCGTGGCGTCGG








TCGTGGTAGATAGGCGGTCATGCATACGAATTTTCAGCTCTTGTTCTG








GTGAC







 


hsa-mir-153-2
AGCGGTGGCCAGTGTCATTTTTGTGATGTTGCAGCTAGTAATATGAGC
7
157059789
157059875
−1
928



CCAGTTGCATAGTCACAAAAGTGATCATTGGAAACTGTG







 


hsa-mir-181b-1
CCTGTGCAGAGATTATTTTTTAAAAGGTCACAATCAACATTCATTGCT
1
197094625
197094734
−1
929



GTCGGTGGGTTGAACTGTGTGGACAAGCTCACTGAACAATGAATGCAA








CTGTGGCCCCGCTT







 


hsa-mir-548h-3
TCTGATTCTGCATGTATTAGGTTGGTGCAAAAGTAATCGCGGTTTTTG
17
13387571
13387688
−1
930



TCATTGAAAGTAATAGCAAAAACTGCAATTACTTTTGCACCAACCTAA








AAGTAGTCACTGTCTTCAGATA







 


hsa-mir-548h-4
GCTATTAGGTTGGTGCAAAAGTAATCGCGGTTTTTGTCATTACTTTAA
8
26962287
26962397
−1
931



TTACTTTACGTTTCATTAATGACAAAAACCGCAATTACTTTTGCACCA








ACCTAATACTTGCTA







 


hsa-mir-5481
TATTAGGTTGGTGCAAAAGTATTTGCGGGTTTTGTCGTAGAAAGTAAT
11
93839309
93839394
−1
932



GGCAAAAACTGCAGTTACTTGTGCACCAACCAAATGCT









5.6 Example 6
Analysis of MicroRNA Count Ratio from smRNASeq Datasets
Table 18 shows the ratio of the number of counts in the sepsis smRNASeq dataset versus the average number of counts from all of the other smRNASeq datasets for each novel microRNA for which the ratio was 2 or higher.







TABLE 18







Novel microRNAs with counts at least 2-fold


higher than average count in other datasets













Counts sepsis/
Ratio counts sepsis/





average count
average count
SEQ ID


microRNA
Rank
other datasets
other datasets
NO














13446-R
1
140/12.81
11
231


13629-L
1
 860/256.28
3
235


13642-R
1
768/55.16
14
236


13661-R
1
47/7.12
7
237


13667-R
1
17/7.4 
2
239


13677-L
1
817/60.33
14
242


13694-L
1
 96/13.75
7
245


13719-L
1
141/29.33
5
253


13729-L
1
143/22.58
6
257


13731-L
1
32/8.36
4
258


14086-L
1
18789/900.97 
21
260


14093-L
1
3327/276.54
12
261


14111-L
1
394/46.66
8
262


14113-L
1
26/4.15
6
263


14177-L
1
4202/280.34
15
266


14371-R
1
 67/12.30
5
269


14482-R
1
16/3.31
5
275


2851-R
1
20/6.33
3
280


6415-R
1
572/43.61
13
287


11626-R
2
140/45.26
3
290


13447-R
2
25/9.8 
3
298


13448-R
2
25/9.8 
3
299


13465-R
2
137/30.62
4
300


13629-R
2
146/51.71
3
302


13640-L
2
225/43.28
5
303


13686-L
2
16/6.88
2
304


13710-R
2
18/6.33
3
307


14327-R
2
39/16.4
2
312


14347-R
2
17/5.1 
3
314


11607-R
3
232/64.96
4
332


13214-L
3
29/9.79
3
335


13244-R
3
 35/10.57
3
336


13452-R
3
 37/17.92
2
338


13677-R
3
116/51.03
2
341


14085-L
3
1777/287.46
6
342


14556-R
3
3397/566.68
6
352


607-R
3
 41/19.70
2
356


14244-L
4
 708/211.84
3
367


373-R
4
443/130.5
3
377


6216-L
4
101/36.51
3
378


4135-R
5
117/35.45
3
399









MicroRNAs 13446-R, 13642-R, 13661-R, 13677-L, 13694-L, 13719-L, 13729-L, 14086-L, 14093-L, 14111-L, 14113-L, 14177-L, 14371-R, 14482-R, 6415-R, 13640-L, 14085-L, and 14556-R (SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, and 352 respectively) had counts in the sepsis smRNASeq dataset of at least 5-fold greater than the average counts in the other datasets. Of those, microRNAs 13446-R, 13642-R, 13677-L, 14086-L, 14093-L, 14177-L, and 6415-R (SEQ ID NOs: 231, 236, 242, 260, 261, 266, and 287, respectively) had counts in the sepsis smRNASeq dataset of at least 10-fold greater than the average counts in the other datasets.
Table 19 shows the ratio of the number of counts in the sepsis smRNASeq dataset versus the average number of counts from all of the other smRNASeq datasets for each microRNA from miRBase for which the ratio was 2 or higher.







TABLE 19







MicroRNAs with counts at least 2-fold higher


than average count in other datasets













Counts sepsis/
Ratio counts sepsis/





average count
average count
SEQ ID


microRNA
Rank
other datasets
other datasets
NO














miR-140-5p
1
13151/840.48 
16
566


miR-142-3p
1
609070/16023.27
38
567


miR-142-5p
1
8409/369.84
23
568


miR-144*
1
47480/3056.12
16
571


miR-144
1
736/38.88
19
570


miR-1537
1
48/6.58
7
573


miR-15a
1
31217/2471.38
13
574


miR-16-1*
1
237/35.03
7
575


miR-185*
1
290/40.12
7
577


miR-2115*
1
199/34.66
6
579


miR-2115
1
57/15.5
4
578


miR-223
1
54934/2708.04
20
580


miR-223*
1
7766/722.27
11
581


miR-451
1
40796/2026.45
20
588


miR-548f
1
848/106.8
8
591


miR-618
1
1830/172.7 
11
598


miR-627
1
3189/209.91
15
601


miR-148a*
2
11446/970.839
12
608


miR-450b-5p
2
1137/131.39
9
612


miR-503
2
2319/271.67
9
613


miR-126
3
10982/5020.08
2
622


miR-140-3p
3
99511/7917.09
13
624


miR-146a
3
114148/11930.10
10
626


miR-146b-5p
3
114217/11737.09
10
629


miR-17*
3
4530/457.59
10
632


miR-199b-5p
3
1879/255.69
7
635


miR-29b-2*
3
143/27.83
5
637


miR-425*
3
1267/226.26
6
641


miR-454*
3
170/30.93
5
642


miR-542-3p
3
3998/545.18
7
644


miR-598
3
2832/491.33
6
648


miR-181a
4
31615/9973.87
3
664


miR-28-3p
5
 3873/1254.94
3
699









MicroRNAs miR-140-5p, miR-142-3p, miR-142-5p, miR-144*, miR-144, miR-1537, miR-15a, miR-16-1*, miR-185*, miR-2115*, miR-223, miR-223*, miR-451, miR-548f, miR-618, miR-627, miR-148a*, miR-450b-5p, miR-503, miR-140-3p, miR-146a, miR-146b-5p, miR-17*, miR-199b-5p, miR-29b-2*, miR-425*, miR-454*, miR-542-3p, and miR-598 (SEQ ID NOs: 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, and 648, respectively) had counts in the sepsis smRNASeq dataset of at least 5-fold greater than the average counts in the other datasets. Of those, microRNAs miR-140-5p, miR-142-3p, miR-142-5p, miR-144*, miR-144, miR-15a, miR-223, miR-223*, miR-451, miR-618, miR-627, miR-148a*, miR-140-3p, miR-146b-5p, and miR-17* (SEQ ID NOs: 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, and 632, respectively) had counts in the sepsis smRNASeq dataset of at least 10-fold greater than the average counts in the other datasets.
All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.
While various specific embodiments have been illustrated and described, it will be appreciated that changes can be made without departing from the spirit and scope of the invention(s).

## Claims

1. A polynucleotide comprising a sequence of at least 17 contiguous nucleotides that is identical or fully complementary to a sequence of at least 17 contiguous nucleotides of SEQ ID NO: 235, wherein the polynucleotide is fewer than 100 nucleotides long, and wherein the polynucleotide comprises a fluorescent dye.

2. The polynucleotide of claim 1, wherein the polynucleotide comprises a sequence of at least 18 contiguous nucleotides that is identical or fully complementary to a sequence of at least 18 contiguous nucleotides of SEQ ID NO: 235.

3. The polynucleotide of claim 1, wherein the polynucleotide comprises a sequence of at least 19 contiguous nucleotides that is identical or fully complementary to a sequence of at least 19 contiguous nucleotides of SEQ ID NO: 235.

4. The polynucleotide of claim 1, wherein the polynucleotide comprises a sequence of at least 20 contiguous nucleotides that is identical or fully complementary to a sequence of at least 20 contiguous nucleotides of SEQ ID NO: 235.

5. The polynucleotide of claim 1, wherein the polynucleotide further comprises a quencher molecule.

6. The polynucleotide of claim 1, wherein the polynucleotide further comprises a sequence that is not identical or complementary to SEQ ID NO: 235.

7. A composition comprising a plurality of uniquely labeled fluorescence resonance energy transfer (FRET) probes, wherein at least one uniquely labeled FRET probe comprises a sequence of at least 8 contiguous nucleotides that is identical or fully complementary to a sequence of at least 8 contiguous nucleotides of SEQ ID NO: 235, and wherein at least one uniquely labeled FRET probe of the plurality of uniquely labeled FRET probes comprises a sequence of at least 8 contiguous nucleotides that is identical or fully complementary to a sequence of at least 8 contiguous nucleotides of SEQ ID NO: 662, wherein each uniquely labeled FRET probe is between 8 and 100 nucleotides long, and wherein each uniquely labeled FRET probe comprises a fluorescent dye and a quencher molecule.

8. A kit comprising a polynucleotide of claim 1.

9. A kit comprising a composition of claim 7.

10. The kit of claim 8, wherein the kit further comprises at least one polymerase.

11. The kit of claim 8, wherein the kit further comprises dNTPs.