# Patent

## Title
MiR-150 for the treatment of blood disorders

## Abstract

The invention provides methods of treating certain blood related disorders, in particular, thrombocytopenia and anemia comprising increasing miR-150 expression or inhibiting miR-150 in progenitor cells respectively.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 13/652,672 filed on Oct. 16, 2012, now U.S. Pat. No. 8,530,443, which is a continuation application of U.S. patent application Ser. No. 12/363,016 filed on Jan. 30, 2009, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/062,931 filed on Jan. 30, 2008, and U.S. Provisional Application No. 61/086,556 filed on Aug. 6, 2008, the contents of each of which are incorporated herein by reference in their entireties.


GOVERNMENT SUPPORT
This invention was made with Government support under Grant No. HL081030 awarded by the National Institute of Health. The Government has certain rights in this invention.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 20, 2013, is named 030258-061193_SequenceListing and is 185,582 bytes in size.
BACKGROUND OF INVENTION
Blood disorders such as thrombocytopenia and anemia affect a significant population, with anemia being the most common disorder of the two. At least 50% of new patients admitted to a hospital's intensive care unit will develop thrombocytopenia during their stay. The development of thrombocytopenia correlates with mortality, longer duration of mechanical ventilation, and an increased need for blood product transfusion.
Anemia occurs when the level of healthy red blood cells (RBCs) in the body becomes too low. RBCs contain hemoglobin, which carries oxygen to the body's tissues. Thus, low levels of healthy RBCs can cause a variety of complications, including fatigue and stress on bodily organs. More than 3 million people in the United States have anemia. Women and people with chronic diseases are at the greatest risk for anemia. A person presents with anemia when the body loses too much blood (such as with heavy periods, certain diseases, and trauma); or the body has problems making red blood cells; or red blood cells break down or die faster than the body can replace them with new ones; or more than one of these problems happen at the same time.
Aplastic anemia occurs when the bone marrow cannot make enough RBCs. This can be due to a viral infection, or exposure to certain toxic chemicals, radiation, or medications (such as antibiotics, antiseizure drugs, or cancer treatments). Some childhood cancers can also cause aplastic anemia, as can certain chronic diseases that affect the ability of the bone marrow to make blood cells. Vitamin B12 and iron deficiencies also contribute to anemia.
Thrombocytopenia is a deficiency of platelets (thrombocytes). The blood usually contains about 140,000 to 440,000 platelets per microliter. Bleeding can occur with relatively minor trauma when the platelet count falls below about 50,000 platelets per microliter of blood. The most serious risk of bleeding, however, generally does not occur until the platelet count falls below 10,000 to 20,000 platelets per microliter. At these very low levels, bleeding may occur without any injury.
Abnormal reductions in the number of platelets are caused when abnormalities occur in any of the following three processes: decreased platelet production by the bone marrow; increased trapping of platelets by the spleen; or a more rapid than normal destruction of platelets. Persons with this condition easily bruise and can have episodes of excess bleeding (a hemorrhage).
Many diseases can cause thrombocytopenia. Thrombocytopenia can occur when the bone marrow does not produce enough platelets, as happens in leukemia, lymphoma and some anemias—aplastic, megaloblastic, vitamin B12 deficiency, and folic acid deficiency. Excessive alcohol consumption can also imped platelet production. Infection with the human immunodeficiency virus (HIV), the virus that causes AIDS, often results in thrombocytopenia. Platelets can become entrapped in an enlarged spleen, as happens in myelofibrosis and Gaucher's disease, reducing the number of platelets in the bloodstream. Massive blood transfusions can dilute the concentration of platelets in the blood. Finally, the body may use or destroy too many platelets, as occurs in many disorders, three of the most notable being idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, and hemolytic-uremic syndrome.
Currently, the treatment options for anemia and thrombocytopenia are directed at the immediate increase of circulating RBC and platelet respectively, followed by identifying the underlying causes. Alternative treatment methods aimed at boosting the innate production of RBCs and platelets, for example, the use of erythropoietin and thrombopoietin to stimulate the bone marrow to produce more red blood cells, are still needed and will be useful in complementing existing treatments for anemia and thrombocytopenia.
SUMMARY OF THE INVENTION
Embodiments of the invention provide methods of treating certain blood related disorders, in particular, thrombocytopenia and anemia. Thrombycytopenia is a condition where there is low platelet count in the blood. Anemia is a condition where there is a low number of red blood cells (RBC) in the blood. Embodiments of the inventions are based on the discovery that miR-150 is involved in the differentiation of megakaryocyte-erythrocyte progenitor cells (MEPs) from the bone marrow. Overexpression of miR-150 can shift more MEPs toward megakaryocyte differentiation and also block erythrocyte maturation. In contrast, a lower level of miR-150 expression shift more MEPs towards erythrocyte differentiation. Accordingly, embodied in the invention is a method of treating thrombocytopenia in a host in need thereof, the method comprising administering to a host an effective amount of an agent that increases miR-150 expression in a cell.
The cell being administered an effective amount of an agent that increases miR-150 expression is a progenitor cell, preferably, a hematopoietic progenitor cell. The increase in miR-150 expression promotes megakaryocyte differentiation from the hematopoietic progenitor cell and consequently more platelets are produced.
In one embodiment, the agent that increases miR-150 expression in a cell comprises a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1. In other embodiments, the nucleic acid is at least 92%, at least 93% at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and all the intermediate percentages between 90% and 100%, identical to SEQ. ID. No. 1. The differences from SEQ. ID. No. 1 should be such that the overall hairpin structure of the pri-miR-150 is maintained. The agent serves to increase the basal level of miR-150 in hematopoietic progenitor cells. The vector can be a virus or a non-virus.
In another embodiment, the agent that increases miR-150 expression in a cell comprises a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1. In other embodiments, the nucleic acid is at least 92%, at least 94%, at least 95%, at least 97%, at least 99%, and all the intermediate percentages between 90% and 100%, identical to SEQ. ID. No. 1. The differences from SEQ. ID. No. 1 should be such that the overall hairpin structure of the pri-miR-150 is maintained.
Embodied herein is a method of treating thrombocytopenia in a host in need thereof, the method comprising: (a) obtaining a sample of hematopoietic progenitor cells from the host; (b) contacting the hematopoietic progenitor cells with a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1; and (c) introducing the cell from step b into the host.
Also embodied herein is a method of treating anemia in a host in need thereof, the method comprising administering to a host an effective amount of an agent that inhibits miR-150 in a cell.
The cell being administered an effective amount of an agent that inhibits miR-150 expression is a progenitor cell, preferably, a hematopoietic progenitor cell. By inhibiting miR-150 expression in the cells, the repression associated with the miR-150 is relieved and erythrocyte differentiation from the progenitor cells is promoted.
In one embodiment, the agent comprises a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 3. In another embodiment, the agent is an antagomir of miR-150, an anti-miR-150 oligonucleotide, an antisense oligonucleotide to miR-150, a locked nucleic acid that anneals to miR-150, or a double strand RNA. Nucleic acid sequences similar to SEQ. ID. No. 3, antagomir of miR-150, an anti-miR-150 oligonucleotide, an antisense oligonucleotide to miR-150, a locked nucleic acid that anneals to miR-150, or a double strand RNA all complementary base-pair with miR-150, although not necessarily perfectly, and can thus inhibit miR-150 from complexing with the miRISC. The vector comprising a nucleic acid sequence can be virus or a non-virus.
Embodied herein is a method of treating anemia in a host in need thereof, the method comprising: (a) obtaining a sample of hematopoietic progenitor cells from said host; (b) contacting the hematopoietic progenitor cells with a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 3; and (c) introducing the cell from step b into the same host.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows the schematic of a novel, sensitive and high-throughput miRNA labeling methodology for expression profiling. RT: reverse transcription; Bi: biotin; arrows: primers.
FIG. 1B shows a heatmap on log 2-transformed data, with black color indicating higher expression and light grey for lower expression. Data reflect the median expression of miRNAs within corresponding populations. Arrows point to miRNAs mentioned in text. Multiple harvests of MEP (n=8), MEGA1 (n=4), MEGA2 (n=6), ERY1 (n=4), ERY2 (n=3), and ERY3 (n=2) populations were purified from human umbilical cord blood cells (23) and profiled for miRNA expression.
FIG. 1C shows the median expression of miR-150 was plotted for each population, with the oval area proportional to the expression level.
FIG. 1D shows miR-150 expression measured using quantitative RT-PCR on multiple harvests of MEP (n=3), MEGA1 (n=4), MEGA2 (n=3), ERY1 (n=3), ERY2 (n=3) and ERY3 (n=3) populations. The ΔCt values (Ct (threshold cycle) of 18S minus Ct of miR-150) are shown for all samples. Note that ΔCt reflects log scale of expression. Samples indicated with black dots were also used in miRNA profiling in FIGS. 1B and 1C, whereas those with open circles were additional samples.
FIG. 2A shows representative flow cytometry plots of the differentiation lineage of CD34+ hematopoietic progenitors cells transduced with constructs expressing a control hairpin (shLuc), miR-150, a mutant miR-150 or miR-15b-16-2. Plots represents lineage markers CD41 (megakaryocytic) and GlyA (erythroid).
FIG. 2B shows the histograms of the data obtained for FIG. 2A. Error bars reflect standard deviation. n=3
FIG. 2C shows the percentage of bone marrow GFP+ or GFP− megakaryocytes (CD41+Ter119−)-J) in mice transduced with either miR-150 and a GFP marker or with control retroviral vector and analyzed 5 to 8 weeks post-transplantation. Each dot represents data from one recipient mouse. n=7.
FIG. 2D shows the percentage of bone marrow GFP+ or GFP− erythrocyte (CD41−Ter119+) in mice transduced with either miR-150 and a GFP marker or with control retroviral vector and analyzed 5 to 8 weeks post-transplantation. Each dot represents data from one recipient mouse. n=7.
FIG. 2E shows representative flow cytometry plots on bone marrow cells with megakaryocytic (CD41) and erythroid (Ter119) markers from data of FIGS. 2C and 2D.
FIG. 2F shows the PF4 expression of recipient bone marrow cells that are GFP+ and GFP−. Each pair of bars represents data from one recipient mouse.
FIG. 2G shows the ratio of GFP+ platelet percentage to the percentage of GFP+ bone marrow cells in the peripheral blood of recipient animals 7-week post-transplantation. T was plotted to reflect the thrombocytogenic potential of bone marrow cells. n=5.
FIG. 2H show the representative flow cytometry plots of bone marrow cells assayed with CD71 and Ter119. The gates R1 to R4 represent immature to mature erythrocytes.
FIG. 2I shows a ratio between GFP+ and GFP− population within the same recipient mouse based upon the percentage of R1 population among all erythrocytes (sum of R1 to R4), n=7. P<0.002.
FIG. 2J shows a ratio between GFP+ and GFP− population within the same recipient mouse based on data for R4 population. P<2×10-4.
FIG. 3A shows the megakaryocyte colony forming units (CFU-Mks) formed from GFP+ and GFP− populations of bone marrow cells isolated from mice transduced with miR-150 retroviral vector or vector control. CFU-Mks were quantitated from 100,000 sorted cells. n=4.
FIG. 3B shows the effects of solvent (PBS), antagomir against miR-150 (anti-150) or a scrambled antagomir on the differentiation of CFU-Mk from MEP cells sorted from wild-type C57BL/6J mice. 4000 cells were analyzed per assay. n=4.
FIG. 3C shows the effects of solvent (PBS), antagomir against miR-150 (anti-150) or a scrambled antagomir on the differentiation of CFU-Mk from Lin-Kit+Sca+ (LKS) stem cells. 1000 cells were analyzed per assay. n=4.
FIG. 3D shows the miR-150 expression in mice were treated with phenylhydrazine to induce anemia, or in mice were treated with PBS (mock). miR-150 expression was assayed by qRT-PCR in lineage negative cells purified from bone marrow. n=3. Error bars represent standard deviation.
FIG. 4A shows the Western blot analysis for MYB and beta-Tubulin expressed in K562 cells transduced with constructs expressing GFP, mutant miR-150 or miR-150.
FIG. 4B shows the design of luciferase reporters for human MYB 3′ UTR, with grey vertical bar indicating wild-type (wt) sites and black indicating mutant (mut) sites. The mutant miRNA site and mutant 3′UTR sites are complementary.
FIG. 4C shows a normalized plot of luciferase activities in 293T cells transduced with constructs expressing GFP, mutant miR-150 or miR-150. Error bars represent standard deviation. n=8.
FIG. 4D shows the histograms of the differentiation lineages of cells transduced with miR-150 and MYB expression constructs and their corresponding vector controls (shLuc, Vector). The lineage markers are CD41 (megakaryocytic) and GlyA (erythroid). n=3. Error bars reflect standard deviation. * P=0.04.
FIG. 4E shows the representative flow cytometry plots of the differentiation lineages in FIG. 4D. Plots represents lineage markers CD41 (megakaryocytic) and GlyA (erythroid).
FIG. 5A shows the reproducibility performance of the plate capture method of miRNA labeling.
FIG. 5B shows the comparison of methods. miRNA expression profiling was performed on the same MCF-7 and 293T total RNA using either the plate capture method, or the previously reported method involving multiple denaturing acrylamide gel purification of small RNAs (“gel purification method”).
FIG. 6A shows the expression of miR-150 in FACS-sorted umbilical cord blood populations. Expression is measured by quantitative RT-PCR analysis as in FIG. 1D were plotted in an oval plot with the oval area proportional to the median value of 2ΔCt for each of the populations.
FIG. 6B shows the histogram of miR-150 expression in CD41+CD61+ and CD71+GlyA+ cells FACS-sorted from the bone marrow of a healthy adult human donor. miR-150 expression was measured using quantitative RT-PCR. 2ΔCt values are shown. Error bars represent standard deviation of measurement.
FIG. 7 shows the evolutionary conservation of mature miR-150 sequences of across multiple species (SEQ ID NOS: 786-791, respectively, in order of appearance). Sequence data were from miRBASE. Big and bold letters indicate non-conserved bases.
FIG. 8 shows the miR-150 expression in cultured human CD34+ bone marrow cells transduced with a control vector (shLuc, n=3) or a m-iR150 construct (n=3), and in multiple harvests of MEP, ERY1, ERY2, ERY3, MEGA1 and MEGA2 populations (as described in FIG. 1D). Threshold cycle values were normalized against 18S ribosomal RNA levels. ΔCt values are plotted
FIG. 9 shows a schematic model for murine bone marrow transplantation.
FIG. 10A shows the gated forward and side scattering flow cytometry analysis of bone marrow cells from wild-type mouse and recipient mice 5-8 weeks after transplantation with vector control or miR-150 vector. GFP gating was determined on cells from wild-type animal.
FIG. 10B shows the GFP gated flow cytometry analysis of bone marrow cells from recipient mice 5-8 weeks after transplantation with vector control.
FIG. 10C shows the GFP gated flow cytometry analysis of bone marrow cells from recipient mice 5-8 weeks after transplantation with miR-150 vector.
FIG. 10D shows the GFP gated flow cytometry analysis of bone marrow cells from wild-type mouse.
FIG. 11A shows the gated sensitized forward and side scattering flow cytometry analysis of platelets in peripheral blood of wild-type mouse and from recipients 7 weeks after transplantation with vector control or miR-150 vector. Peripheral blood cells were stained with CD41 antibody.
FIG. 11B shows the GFP and CD41 flow cytometry analysis of platelets in peripheral blood from recipients 7 weeks after transplantation with vector control.
FIG. 11C shows the GFP and CD41 flow cytometry analysis of platelets in peripheral blood from recipients 7 weeks after transplantation with miR-150 vector.
FIG. 11D shows the GFP and CD41 flow cytometry analysis of platelets in peripheral blood from wild-type mouse.
FIG. 12A shows the absolute cell numbers of GFP+ erythrocytes in the bone marrow of vector control or miR-150 recipient mice. Data reflect cell numbers from two legs of each mouse. Cell number was calculated based on total bone marrow cell yield, GFP status and megakaryocyte and erythrocyte percentage as determined by CD41 and Ter119 staining.
FIG. 12B shows the absolute cell numbers of GFP+ megakaryocytes in the bone marrow of vector control or miR-150 recipient mice.
FIG. 12C shows the absolute cell numbers of GFP− erythrocytes in the bone marrow of vector control or miR-150 recipient mice.
FIG. 12D shows the absolute cell numbers of GFP− megakaryocytes in the bone marrow of vector control or miR-150 recipient mice.
FIG. 13 shows that miR-150 decreases erythroid colony formation.
FIG. 14 shows that antagomir-150 knocks down miR-150 expression.
FIG. 15 shows the formation of megakaryocyte colony in the presence of antagomir-150. Brown color reflects megakaryocyte-specific acetylchol inesterase activity.
FIG. 16A shows the MYB expression as measured using quantitative RT-PCR on multiple harvests of MEP (n=3), MEGA1 (n=4), MEGA2 (n=3), ERY1 (n=3), ERY2 (n=3) and ERY3 (n=3) populations. The ΔCt values (Ct of 18S minus Ct of MYB) are shown for all samples. Samples with black dots were used in miRNA profiling in FIGS. 1B and 1C, whereas those with open circles are additional samples.
FIG. 16B shows the data plotted in an oval plot with the oval area proportional to the median value of 2ΔCt for each of the MEP, MEGA1, MEGA2, ERY1, ERY2 and ERY3 populations.
FIG. 17A shows the human MYB 3′UTR sequence (SEQ ID NO: 785) with four putative miR-150 binding sites in boxes.
FIG. 17B shows the conservation of the four putative miR-150 targeting sites across several shown species (SEQ ID NOS: 792-798, respectively, in order of appearance). Conservation data were obtained from UCSC genome browser.
FIG. 18A shows the knockdown efficiency of two independent shRNAs against MYB expression in MYB-expressing K562 cells. MYB expression was measured by quantitative RT-PCR. The values of 2ΔCt are shown.
FIG. 18B shows the percentage of differentiated megakaryocytes (CD41+GlyA−) from CD34+ human adult bone marrow cells transduced with control vector (shLuc), miR-150 or shRNAs against MYB in an in vitro culture. n=3. Error bars represent standard deviation.



DETAILED DESCRIPTION OF THE INVENTION
Definitions of Terms
As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.
The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
As used herein, the term “therapeutically effective amount” refers to an amount of an agent that is sufficient to effect a therapeutically significant increase in the circulating platelet count in a host diagnosed with thrombocytopenia to at least above 1.6×105 platelets/mm3 or an amount of an agent that is sufficient to effect a therapeutically significant increase in the circulating RBC count in a host diagnosed with anemia to at least above 4.0×1012 red cells/L in adults and 4.6×1012 red cells/L in children.
As used herein, the term “treating thrombocytopenia” refers to a means of increasing the number of circulating platelets in a host who has low platelet count, less than about 1.6×105 platelets/mm3.
As used herein, the term “agent” refers to a nucleic acid sequence or a vector. The nucleic acid sequence can have modifications such as 2′O-methylation and 3′ end cholesterol found in antagomirs and locked nucleic acid oligonucleotides.
As used herein, the term “complementary base pair” refers to A:T and G:C in DNA and A:U in RNA. Most DNA consists of sequences of nucleotide only four nitrogenous bases: base or base adenine (A), thymine (T), guanine (G), and cytosine (C). Together these bases form the genetic alphabet, and long ordered sequences of them contain, in coded form, much of the information present in genes. Most RNA also consists of sequences of only four bases. However, in RNA, thymine is replaced by uracil (U).
As used herein, the term “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one strand nucleic acid of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the template nucleic acid is DNA. In another aspect, the template is RNA. Suitable nucleic acid molecules are DNA, including genomic DNA, ribosomal DNA and cDNA. Other suitable nucleic acid molecules are RNA, including mRNA, rRNA and tRNA. The nucleic acid molecule can be naturally occurring, as in genomic DNA, or it may be synthetic, ie., prepared based up human action, or may be a combination of the two. The nucleic acid molecule can also have certain modification such as 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA), cholesterol addition, and phosphorothioate backbone as described in US Patent Application 20070213292; and certain ribonucleoside that are is linked between the 2′-oxygen and the 4′-carbon atoms with a methylene unit as described in U.S. Pat. No. 6,268,490, wherein both patent and patent application are incorporated hereby reference in their entirety.
The term “vector”, as used herein, refers to a nucleic acid construct designed for delivery to a host cell or transfer between different host cells. As used herein, a vector can be viral or non-viral.
As used herein, the term “expression vector” refers to a vector that has the ability to incorporate and express heterologous nucleic acid fragments in a cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
As used herein, the term “heterologous nucleic acid fragments” refers to nucleic acid sequences that are not naturally occurring in that cell. For example, when a miR-150 gene is inserted into the genome of a bacteria or virus, that miR-150 gene is heterologous to that recipient bacteria or virus because the bacteria and viral genome do not naturally have the miR-150 gene.
As used herein, the term “viral vector” refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the miR-150 gene in place of non-essential viral genes. The vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
The term “replication incompetent” as used herein means the viral vector cannot further replicate and package its genomes. For example, when the cells of a subject are infected with replication incompetent recombinant adeno-associated virus (rAAV) virions, the heterologous (also known as transgene) gene is expressed in the patient's cells, but, the rAAV is replication defective (e.g., lacks accessory genes that encode essential proteins from packaging the virus) and viral particles cannot be formed in the patient's cells.
The term “gene” means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5′ untranslated (5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).
As used herein, “identity”, in the context of two or more nucleic acids sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions, such as when using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.”This term also refers to, or can be applied to, the complement of a test sequence. The term also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 nucleotides in length, or more preferably over a region that is 50-100 nucleotides in length. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443, 1970, by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds. 1995 supplement)).
Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ea., Oxford University Press, New York, 1988; Biocomputing: Informatics and—14 Genome Projects, Smith, D. W., ea., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988)). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs such as BLASTP.
Where necessary or desired, optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482 (1981), which is incorporated by reference herein), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443-53 (1970), which is incorporated by reference herein), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444-48 (1988), which is incorporated by reference herein), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection. (See generally Ausubel et al. (eds.), Current Protocols in Molecular Biology, 4th ed., John Wiley and Sons, New York (1999)).
One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show the percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (J. Mol. Evol. 25:351-60 (1987), which is incorporated by reference herein). The method used is similar to the method described by Higgins and Sharp (Comput. Appl. Biosci. 5:151-53 (1989), which is incorporated by reference herein). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
Another example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described by Altschul et al. (J. Mol. Biol. 215:403-410 (1990), which is incorporated by reference herein). (See also Zhang et al., Nucleic Acid Res. 26:3986-90 (1998); Altschul et al., Nucleic Acid Res. 25:3389-402 (1997), which are incorporated by reference herein). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information internet web site. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990), supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-9 (1992), which is incorporated by reference herein) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77 (1993), which is incorporated by reference herein). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a reference amino acid sequence if the smallest sum probability in a comparison of the test amino acid to the reference amino acid is less than about 0.1, more typically less than about 0.01, and most typically less than about 0.001.
As used herein, the term “a progenitor cell” refers to refer to an immature or undifferentiated cell that has the potential later on to mature (differentiate) into a specific cell type, for example, a blood cell, a skin cell, a bone cell, or a hair cells. A progenitor cell also can proliferate to make more progenitor cells that are similarly immature or undifferentiated.
As used herein, the term “hematopoietic progenitor cell” refers to progenitor cells that can differentiate into the hematopoietic lineage and give rise to all blood cell types such as white blood cells and red blood cells.
As used herein, the term “microRNA or miRNA” refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation. See, e.g., Carrington and Ambros, 2003, Science, 301(5631):336-8 which is hereby incorporated by reference in its entirety. miRNA are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA); instead they are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression. The term will be used to refer to the RNA molecule processed from a precursor pre-miRNA.
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in hematology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 18th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R Kimmerl Eds., Academic Press Inc., San Diego, USA (1987)); Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley and Sons, Inc.); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.); Current Protocols in Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons, Inc.); Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.); Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005); Animal Cell Culture Methods (Methods in Cell Biology, Vol 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.
It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and, as such, may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages may mean±1%.
The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term “comprises” means “includes.” The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”
All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
Embodiments of the Inventions
Embodiments of the methods disclosed herein are based on the discovery that a microRNA (miRNAs), miR-150, regulates the differentiation of megakaryocyte-erythrocyte progenitor cells (MEPs) from the bone marrow. The regulation of the developmental fate of multi-potential cells is not well known and the process by which bipotential hematopoietic progenitor cells are driven to become either red blood cells or platelets is not understood. The inventors discovered that miRNAs control mammalian cell fate of the multi-potential MEPs, in particular, miR-150 modulates lineage fate in MEPs. The inventors found that miR-150 is preferentially expressed in the megakaryocytic lineage and that miR-150 expression in MEPs drives MEP differentiation toward megakaryocytes at the expense of erythroid cells in vitro and in vivo.
In experiments using human bone marrow hematopoietic progenitor cells, overexpression of the miR-150 gene resulted in a significant increase in megakaryocyte colony forming units (CFU-Mk) differentiating from MEPs with a concomitant decrease in erythroid colony forming units. When similar miR-150 overexpression experiments were conducted with murine bone marrow hematopoietic progenitor cells and the resultant miR-150 overexpressing progenitor cells were transplanted back into the animal, the mice exhibited a larger population of megakaryocytes and a greater number of platelets compared to control mice miR-150 expression led to a bona fide increase in bone marrow megakaryocytes that were competent to produce mature platelets in circulation. MiR-150 expression induces a blockage in the earliest definable stage of erythropoiesis, in addition to causing a significant reduction in the total erythroid population. Accordingly, an increase amount of miR-150 regulates the differentiation of MEPs and the post-commitment megakaryocyte expansion.
While overexpression of miR-150 leads to pro-megakaryocyte differentiation and an increase in platelet count, the inhibition of miR-150 expression results in more erythroid colony forming units, more erythrocytes production and a corresponding decrease in CFU-Mk. In an artificial model of induced anemia, an inhibition of miR-150 with a specific antagomir elevated the erythrocyte count in the model animal. Clearly, one of the roles of miR-150 is in vivo is to regulate the differentiation of bi-potential MEPs and the production of RBCs and platelets.
MicroRNAs (miRNAs) are a class of 18-24 nt non-coding RNAs (ncRNAs) that exist in a variety of organisms, including mammals, and are conserved in evolution. miRNAs are transcribed as 5′-capped large polyadenylated transcripts (pri-miRNA), primarily in a Pol II-dependent manner Approximately 40% of human miRNAs are co-transcribed as clusters encoding up to eight distinct miRNA sequences in a single pri-microRNA transcript. Many miRNAs can be encoded in intergenic regions, hosted within introns of pre-mRNAs or within ncRNA genes. Many miRNAs also tend to be clustered and transcribed as polycistrons and often have similar spatial temporal expression patterns. MiRNAs have been found to have roles in a variety of biological processes including developmental timing, differentiation, apoptosis, cell proliferation, organ development, and metabolism (Kloosterman, et al., 2006 Dev Cell 11:441-50, and Krutzfeldt, et al., 2006 Cell Metab 4:9-12). Furthermore, miRNAs have been implicated in diseases such as cancer (Esquela-Kerscher, et al., 2006 Nat Rev Cancer 6:259-69) and hepatitis C (Jopling, et al., 2005 Science 309:1577-81), which make them attractive new drug targets. In contrast to the widely used RNAi technology using small interfering RNA (siRNA) duplexes, strategies to inhibit miRNAs have been less well investigated. Reverse-complement 2′-O-methyl sugar modified RNA is frequently being used to block miRNA function in cell-based systems (Krutzfeldt, et al., 2006 Nat Genet. 38:S14-9).
Pri-miRNAs are cleaved within the nucleus by the microprocessor complex consisting of Drosha, an RNaseIII-type nuclease and a protein co-factor, DGCR8 (DiGeorge syndrome critical region 8 gene) in humans, Pasha in Drosophila. The resulting 60-70 nucleotide hairpin structure (pre-miRNA) encodes for a single miRNA sequence that is exported from the nucleus to the cytoplasm by Exportin5 in a Ran-GTP dependent manner Cytoplasmic pre-miRNAs are further cleaved, by another RNaseIII-nuclease, Dicer in concert with cofactors (TRBP and PACT in humans), to remove the loop sequence forming a short-lived asymmetric duplex intermediate (miRNA: miRNA *). The microRNA: microRNA * duplex is in turn loaded into the miRISC complex in which Argonaut (Ago) proteins appear to be the key effector molecules. The strand that becomes the active mature microRNA appears to be dependent upon which has the lowest free energy 5′ end and the other strand is degraded by an unknown nuclease.
Accordingly, in one embodiment, disclosed herein is a method of treating thrombocytopenia in a host in need thereof, the method comprising administering to a host an effective amount of an agent that increases miR-150 expression in a cell.
Thrombocytopenia is a deficiency of platelets (thrombocytes). Platelets come from megakaryocytes, which are produced in the bone marrow from hematopoietic progenitor cells. When abnormalities develop in the marrow, the marrow cells can lose their ability to produce platelets in correct amounts. The result is a lower than normal level of platelets in the blood. Drugs used in cancer chemotherapy can cause the marrow to malfunction in this way, as can the presence of tumor cells in the marrow itself.
Normally, the spleen holds about one-third of the body's platelets as part of this organ's function to recycle aging or damaged RBCs. When liver disease or cancer of the spleen is present, the spleen can enlarge, resulting in a greater number of platelets staying in the organ. This condition then results in abnormally low numbers of platelets in the blood.
Platelets can break down in unusually high amounts in persons with abnormalities in their blood vessel walls; with blood clots; or with man-made replacement heart valves. Devices placed inside blood vessels to keep them from closing (stents) due to weakened walls or fat build-up can also cause platelets to break down. In addition, infections and other changes in the immune system can speed up the removal of platelets from the circulation.
Thrombocytopenia generally means a circulating platelet count of less than the normal circulating range, e.g., less than about 1.6×105/mm3, less than about 1.5×105/mm3, less than about 1.3×105/mm3 or less than about 1.0×105/mm3. Under the common terminology criteria for adverse events, version 3.0, grade 1 thrombocytopenia is the lower normal limit to 75,000 platelets/mm3, grade 2 thrombocytopenia is <75,000-50,000 platelets/mm3, grade 3 thrombocytopenia is <50,000-25,000 platelets/mm3 and grade 4 is <25,000 platelets/mm3.
The host needing treatment for thrombocytopenia can be any animal that has platelets, the platelets are produced from megakaryocytes, and the megakaryocytes are differentiated from hematopoietic progenitor cells. In one embodiment, the host is a mammal, such as a dog, cat, horse, and monkey, preferably a human.
A platelet count is performed to determine the number of platelets in circulation and on the basis of the platelet counts, the physician can determine whether the host has thrombocytopenia. A platelet count is part of the complete blood count test (CBC) routinely ordered by physicians. A platelet count is a test to measure platelets that are present in the peripheral circulating blood of a host. This test can be performed by skilled medical personnel such as physicians, nurses and trained laboratory technicians by methods known in the art. For example, a sample of peripheral blood is drawn into anticoagulant to prevent the blood from clotting. The larger and heavier cells: white blood cells and RBCs are sedimented by low speed centrifugation (1000×G, 10 min) and the platelet-rich liquid fraction of the blood is collected and counted. Other manual methods of determining platelet counts include visual evaluation of blood smears on microscope slides and methods using RBCs-lysing agents followed by visual platelet counting. In one embodiment, platelets are determined using automated blood counting machines that include but are not limited to the Sysmex XE-2100, the Abbott Cell-Dyn range (e.g. Cell-Dyn 3500), Boule Nordic AB Ca530 Vet and Melet Schloesing MS4. In one embodiment, the platelet count is performed according to the European Patent EP1123510 and U.S. Pat. No. 6,872,572, both of which are hereby incorporated by reference in their entirety.
In one embodiment, the cell from a host needing treatment for thrombocytopenia wherein the miR-150 expression is increased is a progenitor cell. In another embodiment, the progenitor cell is a hematopoietic progenitor cell.
In one aspect, an agent that increases the miR-150 expression in a cell of a host comprises a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1. In other aspects, the nucleic acid is at least 92%, at least 94%, at least 95%, at least 97%, at least 99%, and all the intermediate percentages between 90% and 100%, identical to SEQ. ID. No. 1. The differences from SEQ. ID. No. 1 should be such that the overall hairpin structure of the pri-miR-150 is maintain.
In another aspect, the vector is a virus. In yet another aspect, the vector is a non-viral vector.
In one embodiment, an agent that increases miR-150 expression in a cell of a host comprises a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1. In another embodiment, the nucleic acid is at least 92%, at least 94%, at least 95%, at least 97%, at least 99%, and all the intermediate percentages between 90% and 100%, identical to SEQ. ID. No. 1.
The SEQ. ID. No. 1 provides the Homo sapiens miR-150 stem-loop pri-miRNA which is also known as has-mir-150, MI0000479, NT—011109, or miRBase:MI0000479 at the miRBase at the Sanger Institute (world wide web “period” microRNA “period” Sanger “period” ac “period” uk).
While not wishing to be bound by theory, expression of the miR-150 gene or a nucleic acid that is at least 90% identical to SEQ. ID. No. 1 by gene transcription produces a primary transcript, pre-miR-150, that can fold into a stem-loop structure. The pre-miR-150 can be exported out of the nucleus and be processed in the cytoplasm into a duplex miR-150 from which a mature miR-150 (SEQ. ID. 2) (miRBase:MIMAT0000451) becomes available for upload into the miRISC (the miRNA gene inhibition complex). A vector comprising a nucleic acid that is at least 90% identical to SEQ. ID. No. 1, when transfected into a host cell, introduces the miR-150 gene as a transgene into the host cell. In this transfected host cell harboring the miR1-50 transgene, overexpression of the miR-150 transgene increases the amount of miR-150 in the cell. Excess amounts of miR-150 can lead to enhanced repression of genes naturally regulated by miR-150.
In one embodiment, a vector comprising a nucleic acid that is at least 90% identical to SEQ. ID. No. 1 is an expression vector. The expression vector can have a strong promoter sequence driving the robust mammalian transcription of the miR-150 transgene in the host cell. Strong promoter sequences include but are not limited to the Moloney murine leukemia virus promoter, cytomegalovirus promoter, the simian virus 40 early region promoter, the lymphotropic papovavirus, and the human beta-globin gene promoter sequences. In one embodiment, the promoter can be chimeric sequences from several promoter types as described in U.S. Pat. No. 6,136,536 which is incorporated hereby reference in its entirety. In another embodiment, the promoter can be the human osteocalcin (hOC) promoter (McCarthy H. O., et. al., 2007, J. Gene Medicine, 9: 511-20).
In one embodiment, the expression vector can be a virus such as an adenovirus, an adeno-associated virus, or lentivirus, for example, MDH.xdna murine retroviral vector. Viral vectors provide an additional advantage of ease of transfecting the host cell by viral infection. In another embodiment, the expression in a non-viral vector. Such vectors can be transfected into host cells using known transfection methods known in the art, such as cationic lipid transfection.
By increasing the miR-150 expression in the hematopoietic progenitor cells of a host, the differentiation of the hematopoietic progenitor cells can be shifted to producing more megakaryocytes, from which platelets are derived, eventually increasing the platelet count.
In one embodiment, disclosed herein is a method of treating thrombocytopenia in a host in need thereof, comprises: (a) contacting the hematopoietic progenitor cells with a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1; and (b) introducing the cell carrying the transgene into the host.
The method comprises obtaining a sample of hematopoietic progenitor cells from a host.
In one embodiment, the hematopoietic progenitor cells are isolated from host, transfected, cultured, and transplanted back into the same host, i.e. an autologous cell transplant. In another embodiment, the hematopoietic progenitor cells are isolated from a donor who is an HLA-type match with a host (recipient) who is diagnosed with thrombocytopenia. Donor-recipient antigen type-matching is well known in the art. The HLA-types include HLA-A, HLA-B, HLA-C, and HLA-D. These represent the minimum number of cell surface antigen matching required for transplantation. The donor's hematopoietic progenitor cells can be transfected with a vector or nucleic acid comprising the nucleic acid that is at least 90% identical to SEQ. ID. No. 1 (the miR-150 transgene), culture expanded, and then transplanted into the host.
In one embodiment, the method disclosed herein includes monitoring the platelet count of a host before and after the administration of the agent for treatment of thrombocytopenia. The platelet count performed before treatment provides the data for a physician to make a diagnosis of thrombocytopenia and the platelet count also serves a reference number from which after the treatment platelet counts can be compared. Routine platelet count of samples of peripheral blood should be performed at 1, 2, 3 months or every bi-monthly after treatment or according to physician's decision in order to monitor the efficacy of the treatment.
In one embodiment, disclosed herein is a method of treating anemia in a host in need thereof, the method comprising administering an effective amount of an agent that inhibits miR-150 expression in a cell to a host.
Anemia generally means a red cell mass corresponding to less than about 4.0×1012 red cells/L in adult females and less than about 4.5×1012 red cells/L in adult males (a hemoglobin level of less than about 12.0 g/dL in adult females and less than about 13.5 g/dL in adult males). Anemia may occur as a result of bleeding (including internal), hemolysis, kidney disease, leukemia, multiple myeloma, bone marrow failure, erythropoietin deficiency, or deficiencies in iron, folate, vitamin B12, or vitamin B6.
The RBCs count is also a part of the complete blood count test (CBC) routinely ordered by physicians. A sample of peripheral blood can be collected and mixed with anticoagulant. For RBC counting by the manual visual method, a small, fixed volume of blood is diluted, applied to a hemacytometer and counted under a microscope. Alternatively, RBCs are counted with automated cell counters described herein.
In one embodiment, a host needing treatment for anemia can be any animal that has RBCs (erythrocytes), and the RBCs are differentiated from hematopoietic progenitor cells. In one embodiment, the host is a mammal, such as a dog, cat, horse, and monkey, preferably a human.
In one embodiment, the cell in a host wherein the miR-150 activity is inhibited, is a progenitor cell. In another embodiment, a progenitor cell wherein the miR-150 activity is inhibited is a hematopoietic progenitor cell.
In one embodiment, an agent that inhibits miR-150 activity in a cell comprises a nucleic acid sequence that can form complementary base-pairing with SEQ. ID. No. 2, the mature miR-150, for at least 90% of the bases of SEQ. ID. No. 2. In one aspect, the nucleic acid can form complementary base-pairing with at least 92%, at least 94%, at least 95%, at least 97%, at least 99%, and all the intermediate percentages between 90% and 100%, to SEQ. ID. No. 2. In another embodiment, an agent is a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 3 (miRBase:MIMAT0004610). The nucleic acid is at least 92%, at least 94%, at least 95%, at least 97%, at least 99%, and all the intermediate percentages between 90% and 100%, identical to SEQ. ID. No. 3. In yet another embodiment, an agent is a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 3.
In some aspects, an agent that inhibits miR-150 activity in a cell can be referred to as a miR-150 inhibitor, the miR-150 inhibitor functions by blocking, preventing, and/or antagonizing the normal cellular activity of the mature miR-150 which is to down regulate the expressions of certain genes. A miR-150 inhibitor can be an antagomir of miR-150, an antisense oligonucleotide to miR-150, a locked nucleic acid that anneals to miR-150, and double-stranded RNA corresponding to miR-150 (dsRNA).
In one embodiment, a miR-150 inhibitor is between 17 and 25 nucleotides in length and that comprises a 5′ to 3′ sequence that is at least 90% complementary to the 5′ to 3′ sequence of SEQ. ID. No. 2. In another embodiment, a miR-150 inhibitor is a synthetic RNA molecule of between 17 and 125 residues in length comprising i) an miRNA region whose sequence from 5′ to 3′ is identical to a mature miR-150 sequence, and ii) a complementary region whose sequence from 5′ to 3′ is between 60% and 100% complementary to the mature miR-150 sequence.
Antagomirs are a novel class of chemically engineered oligonucleotides that block the activity of miRNAs and essentially “silence” the miRNA (Krützfeldt J, et. al., 2005, Nature 438: 685-9). Antagomirs are single-stranded RNA that are perfectly complementary to their miRNA except that they are 2′-O-methyl (2′-OMe) oligoribonucleotides and are also linked to cholesterol at the 3′ end. Both these modifications, 2′-OMe and cholesterol, aid in the antagomir stability in vivo and ease of entry into the cells. Methods of designing and synthesizing antagomirs and the various modifications (e.g. 2′-O-Methoxyethyl) are described in US Pat. Application 20070213292 and is hereby incorporated by reference in its entirety. An example of a miR-150 antagomir is 5′mC(*)mA(*)mCmUmGmGmUmAmCmAmAmGmGmGmUmUmGmGmG(*)mA(*) mG (*)mA (*)(3′-Chl) 3′ (SEQ. ID. No. 23). The mN: 2′OMe base; *: phosphorothioate linkage; Chl: cholesterol.
In one embodiment, the miR-150 inhibitor is a miR-150 antagomir. In one embodiment, the miR-150 inhibitor is SEQ. ID. No. 23. In another embodiment, the miR-150 inhibitor consist essentially of SEQ. ID. No. 23. In another embodiment, the miR-150 inhibitor consist of SEQ. ID. No. 23. In another embodiment, the miR-150 inhibitor comprises SEQ. ID. No. 23.
Locked nucleic acid (LNA)-modified oligonucleotides are distinctive 2′-O-modified RNA in which the 2′-O-oxygen is bridged to the 4′-position via a methylene linker to form a rigid bicycle, locked into a C3′-endo (RNA) sugar conformation (Venter B., et. al., Biochemistry 2004; 43: 13233-13241). The LNA modification leads to the thermodynamically strongest duplex formation with complementary RNA known. Consequently, a biological activity is often attained with very short LNA oligonucleotides. For example, an 8 nt fully-modified LNA oligomer complementary to a structural loop inhibited 50% of self-splicing of group I introns from rRNA genes in pathogenic organisms whereas DNA and RNA oligonucleotides were ineffective. Short fully-modified LNA oligonucleotides designed against telomerase were active in cellular assays, compared to mismatched negative controls. Furthermore, LNAs display excellent mismatch discrimination. Mouritzen et al. (Expert Rev Mol Diagn 2003; 3: 27-38) showed single-nucleotide specificity against complementary DNA using fully modified 12 nucleotide LNA probes coupled to glass slides during the development of a microarray used to probe samples for single-nucleotide polymorphisms (SNPs) associated with human dysmetabolic syndrome. The synthesis and incorporation of LNA bases can be achieved by using standard DNA synthesis chemistry and described in U.S. Pat. No. 6,268,490 and is hereby incorporated by reference in its entirety.
An anti-sense oligonucleotide of miR-150 has a sequence that perfectly complementary to SEQ. ID. No. 2, the mature miR-150. Complementary pairing between an anti-sense oligonucleotide of miR-150 and miR-150 produces a duplex RNA that is highly susceptible to RNase degradation. An anti-sense oligonucleotide of miR-150 comprises the sequence 5′-CACUGGUACAAGGGUUGGGAGA-3′ (SEQ. ID. No. 4).
One skilled in the art can also readily determine an appropriate dosage regimen for administering a compound that inhibits miRNA expression to a given subject, as described herein. Suitable compounds for inhibiting miRNA gene expression include double-stranded RNA (such as short- or small-interfering RNA or “siRNA”), antisense nucleic acids, and enzymatic RNA molecules, such as ribozymes. Each of these compounds can be targeted to a given miRNA gene product and interfere with the expression (e.g., by inhibiting translation, by inducing cleavage and/or degradation) of the target miRNA gene product. For example, expression of a given miRNA gene can be inhibited by inducing RNA interference of the miRNA gene with an isolated double-stranded RNA (“dsRNA”) molecule which has at least 90%, for example at least 95%, at least 98%, at least 99%, or 100%, sequence homology with at least a portion of the miRNA gene product. In a particular embodiment, the dsRNA molecule is a “short or small interfering RNA” or “siRNA.” siRNA useful in the present methods comprise short double-stranded RNA from about 17 nucleotides to about 29 nucleotides in length, preferably from about 19 to about 25 nucleotides in length. The siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions (hereinafter “base-paired”)—The sense strand comprises a nucleic acid sequence that is substantially identical to a nucleic acid sequence contained within the target miRNA gene product.
In one embodiment, an agent that inhibits miR-150 activity is a vector that comprises an anti-sense oligonucleotide to miR-150 (SEQ. ID. No. 4). The anti-sense oligonucleotide sequence can be cloned into a vector for the expression in a host cell by any means known to one skilled in the art. In one embodiment, the vector is a virus. In another embodiment, the vector is a non-virus. Designing, cloning, transfection, and expression of anti-sense oligonucleotides against miRNAs are described in Scherr M. et. al., 2007, Nucleic Acid Research 35(22):e149 and is incorporated hereby reference in its entirety.
In one embodiment, the agent can be various combinations of an antagomir of miR-150, an antisense oligonucleotide to miR-150, dsRNA to miR-150, or a locked nucleic acid that anneals to miR-150.
In one embodiment, disclosed herein is a method of treating anemia in a host in need thereof, the method comprising: (a) contacting the hematopoietic progenitor cells with a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 3 or 4; and (b) introducing the cell from step b into the same host. The method comprises obtaining a sample of hematopoietic progenitor cells from a host. Methods of isolating, transfecting, culturing, screening for strong expression of transgene, and transplantation can be performed as described herein.
In one embodiment, the method disclosed herein includes monitoring the RBC count of a host before and after the administration of the agent for treatment of anemia. The RBC count performed before treatment provide the data for a physician to make a diagnosis of anemia and the RBC count also serve a reference number from which after treatment RBC counts can be compared with. Routine RBC count of samples of peripheral blood should be performed at 1, 2, 3 months or every bi-monthly after treatment or according to physician's decision in order to monitor the efficacy of the treatment.
In one embodiment, the method disclosed herein comprises treating anemia in conjunction with other known treatments such as with erythropoietin (EPO) and peptide mimetics of EPO. EPO is a hormone produced by the kidney that promotes the formation of red blood cells in the bone marrow. The kidney cells that make EPO are specialized and are sensitive to low oxygen levels in the blood. These cells release EPO when the oxygen level is low in the kidney. EPO then stimulates the bone marrow to produce more red cells and thereby increase the oxygen-carrying capacity of the blood. EPO is the prime regulator of red blood cell production. Its major functions are to promote the differentiation and development of red blood cells and to initiate the production of hemoglobin, the molecule within red cells that transports oxygen.
In one embodiment, the methods described herein can be implemented with other therapeutics associated with thrombocytopenia and anemia.
The present invention can be defined in any of the following alphabetized paragraphs:

    
    
        [A] The use of an agent that increases miR-150 expression in a cell for the treatment of thrombocytopenia in a host in need thereof.
        [B] The use of an agent that increases miR-150 expression in a cell in the manufacture of a medicament for the treatment of thrombocytopenia.
        [C] The use of paragraph [A] or [B], wherein the cell is a progenitor cell.
        [D] The use of paragraph [C], wherein the progenitor cell is a hematopoietic progenitor cell.
        [E] The use of paragraph [A] or [B], wherein the agent is a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1.
        [F] The use of paragraph [E], wherein the vector is a virus.
        [G] The use of paragraph [A] or [B], wherein the agent is a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1.
        [H] A method of treating thrombocytopenia in a host in need thereof, the method comprising administering an effective amount of an agent that increases miR-150 expression in a cell to a host.
        [I] The method of paragraph [H], wherein the cell is a progenitor cell.
        [J] The method of paragraph [I], wherein the progenitor cell is a hematopoietic progenitor cell.
        [K] The method of paragraph [H], wherein the agent is a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1.
        [L] The method of paragraph [K], wherein the vector is a virus.
        [M] The method of paragraph [H], wherein the agent is a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1.
        [N] A method of treating thrombocytopenia in a host in need thereof, the method comprising:
        
            a. obtaining a sample of hematopoietic progenitor cells from said host;
            b. contacting the hematopoietic progenitor cells with a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1; and
            c. introducing the cell from step b into the host.
        
        
        [O] The use of an agent that inhibits miR-150 in a cell for the treatment of anemia in a host in need thereof.
        [P] The use of an agent that inhibits miR-150 in a cell in the manufacture of a medicament for the treatment of anemia.
        [Q] The use of either paragraph [O] or [P], wherein the cell is a progenitor cell.
        [R] The use of paragraph [Q], wherein the progenitor cell is a hematopoietic progenitor cell.
        [S] The use of paragraph [O] or [P], wherein the agent is an antagomir of miR-150, an anti-miR-150 oligonucleotide, an antisense oligonucleotide to miR-150 or a locked nucleic acid that anneals to miR-150.
        [T] The use of paragraph [O] or [P], wherein the agent is a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 3.
        [U] The use of paragraph [T], wherein the vector is a virus.
        [V] A method of treating anemia in a host in need thereof, the method comprising administering an effective amount of an agent that inhibits miR-150 in a cell to a host.
        [W] The method of paragraph [V], wherein the cell is a progenitor cell.
        [X] The method of paragraph [W], wherein the progenitor cell is a hematopoietic progenitor cell.
        [Y] The method of paragraph [V], wherein the agent is a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 3.
        [Z] The method of paragraph [V], wherein the agent is an antagomir of miR-150, an anti-miR-150 oligonucleotide, an antisense oligonucleotide to miR-150 or a locked nucleic acid that anneals to miR-150.
        [AA] A method of paragraph [Y], wherein the vector is a virus.
        [BB] A method of treating anemia in a host in need thereof, the method comprising:
        
            a. obtaining a sample of hematopoietic progenitor cells from said host;
            b. contacting the hematopoietic progenitor cells with a vector comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 3; and
            c. introducing the cell from step b into the same host.

Hematopoietic Progenitor Cells

        
        
    
    


Peripheral blood progenitor cells (PBPC) have become the preferred source of hematopoetic progenitor cells for allogeneic and autologous transplantation because of technical ease of collection and shorter time required for engraftment. Traditionally, granulocyte-colony stimulating factor (G-CSF) has been used to stimulate more PBPC and release of hematopoetic progenitor cells from the bone marrow. Although regimens using G-CSF usually succeed in collecting adequate numbers of PBPC from healthy donors, 5%-10% will mobilize stem cells poorly and may require multiple large volume apheresis or bone marrow harvesting.
AMD3100, is a bicyclam compound that inhibits the binding of stromal cell derived factor-1 (SDF-1) to its cognate receptor CXCR4. CXCR4 is present on CD34+ hematopoetic progenitor cells and its interaction with SDF-1 plays a pivotal role in the homing of CD34+ cells in the bone marrow Inhibition of the CXCR4-SDF1 axis by AMD3100 releases CD34+ cells into the circulation, which can then be collected easily by apheresis. Recently, a published report demonstrated that large numbers of CD34+ cells were rapidly mobilized in healthy volunteers following a single subcutaneous injection of AMD3100.
The hematopoietic progenitor cells can be isolated fresh and frozen mononuclear cells of peripheral blood, cord blood, and bone marrow using its pan-hematopoietic antigen CD34 or by other methods that are known to one skilled in the art. For example, antibodies against CD34 can be used for immuno-isolating the CD34(+) hematopoietic progenitor cells from the mononuclear cell fraction. The anti-CD34 antibodies can be conjugated with fluorophores or to magnetic beads for ease of separation by FACS or magnets respectively.
Hematopoietic progenitor cells bearing the pan-hematopoietic antigen CD34 can also be isolated by using taking advantage of the cells ability to bind galactose-conjugated proteins. This lectin-positive sub-population represents approximately 0.1 to 0.5% of the total bone marrow cells, and contains 100% of the hematopoietic progenitor cells. The galactose-binding lectin on these cells is specific for this sugar. Additionally, highly proliferative hematopoietic progenitor cells with very primitive phenotypes, including a newly identified progenitor cell that produces multiple lineages, express this lectin. (Pipia and Long, Nature Biotechnology 15, 1007-1011 (1997)).
In vitro transfection of isolated hematopoietic progenitor cells from a host facilitates targeted transfection of the miR-150 transgene into specific progenitor cells. Transfection of progenitor cells can be accomplished by any transfection methods known in the art, for example, calcium phosphate-mediated, DEAE-Dextran-mediated, calcium alginate microbeads, cation lipid-mediated, scrape-loading, and ballistic bombardment of nucleic acid gold particles. In one embodiment, isolation and culturing of progenitor cells is performed using the methods well known in to those skilled in the art, e.g. as described in U.S. Pat. Nos. 5,199,942, 5,474,687, 5,589,368, 5,612,211, 5,905,041, 6,355,237, and 7,345,025, which are hereby incorporated by reference in their entirety. The identity of the isolated hematopoietic progenitor cells can be confirmed by transglutaminase expression in culture as described in WO2000/006766, which is also hereby incorporated by reference in its entirety. After in vitro transfection, the miR-150 transfection level can be monitored by quantitative real-time PCR with specific primer pairs to the pre-miR-150 and the mature miR-150. The transfected progenitor cells carrying the transgene can be expanded in culture according to methods described in U.S. Pat. Nos. 5,744,361, 5,905,041, and 6,326,198, which are hereby incorporated by reference in their entirety. The expanded progenitor cells with the miR-150 transgene can then be transplanted back into the original host. Transplantation of progenitor cells are described in U.S. Pat. Nos. 5,817,773, 5,858,782, and U.S. patent application Ser. No. 10/730,334 and they are hereby incorporated by reference in their entirety.
In one embodiment, the SEQ. ID. No. 1 (miR-150 gene) is cloned into the MDH.xdna murine retroviral vector and miR-150 retroviral vectors can be transfected into isolated hematopoietic progenitor cells. Forty-eight hours after transfection, total RNAs were isolated and loaded onto a 10% denaturing polyacrylamide gel. DNA oligo probes that were complementary to each of the selected miRNAs were labeled and hybridized to the membrane to detect mature miR-150s that can be efficiently processed (20- to 24-nt). Constructs with high processing efficiency can be selected for bone marrow transplantation.
Expression Vectors and Expression Systems for Expression
Isolated nucleic acid sequences that are at least 90% identical to SEQ. ID. No. 1, 3 and 4 can be obtained using a number of standard techniques. For example, the nucleic acids can be chemically synthesized or recombinantly produced using methods known in the art. In one embodiment, the nucleic acids are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., U.S.A.), Pierce Chemical (part of Perbio Science, Rockford, Ill., U.S.A.), Glen Research (Sterling, Va., U.S.A.), ChemGenes (Ashland, Mass., U.S.A.) and Cruachem (Glasgow, UK).
Alternatively, the nucleic acids and their complementary strands can be synthesized as single strand DNA initially and then subsequently anneal together to form duplex for cloning into vectors for gene expression as described in Scheer M. et. al. supra. Restriction enzyme sites can be designed and incorporated at the ends of the eventual duplex to facilitate ligating the duplex into a vector.
The human miR-150 stem loop (hsa-miR-150) is contained within a 473 bp genomic fragment that includes the hairpin region of hsa-miR-150 and  ̃200 bp of flanking sequence on each side. This genomic expression cassette can be PCR amplified from human genomic DNA (Roche Applied Science) with primers containing 5′ linker sequences harboring relevant digestion sites (core primer sequences: 5′ CAGCATAGGGTGGAGTGGGT3′ (Seq. ID. No. 5); 5′TACTTTGCGCATCACACAGA3′ (SEQ. ID. No. 6).
Once ligated into a vector, the nucleic acid can be subcloned into several expression vectors, such as a viral expression vector or a mammalian expression vector by PCR cloning, restriction digestion followed by ligation, or recombination reaction such as those of the lambda phage-based site-specific recombination using the GATEWAY® LR and BP CLONASETM enzyme mixtures. Subcloning should be unidirectional such that the 5′ transcription start nucleotide of the nuclei acid sequence is downstream of the promoter in the expression vector. Alternatively, when the nucleic acid sequence is cloned into pENTR/D-TOPO®, pENTR/SD/D-TOPO® (directional entry vectors), or any of the INVITROGEN's GATEWAY® Technology pENTR (entry) vectors, the nucleic acid sequence can be transferred into the various GATEWAY® expression vectors (destination) for protein expression in host cells in one single recombination reaction. Some of the GATEWAY® destination vectors are designed for the constructions of baculovirus, adenovirus, adeno-associated virus (AAV), retrovirus, and lentiviruses, which upon infecting their respective host cells, facilitating ease of introducing the transgene into the host cells. The GATEWAY® Technology uses lambda phage-based site-specific recombination instead of restriction endonuclease and ligase to insert a gene of interest into an expression vector. The DNA recombination sequences (attL, attR, attB, and attP) and the LR and BP CLONASETM enzyme mixtures that mediate the lambda recombination reactions are the foundation of GATEWAY® Technology. Transferring a gene into a destination vector is accomplished in just two steps: Step 1: Clone the nucleic acid sequence of interest into an entry vector such as pENTR/D-TOPO®, Step 2: Mix the entry clone containing the nucleic acid sequence of interest in vitro with the appropriate GATEWAY® expression vector (destination vector) and GATEWAY® LR CLONASETM enzyme mix. There are GATEWAY® expression vectors for protein expression in E. coli, insect cells, mammalian cells, and yeast. Site-specific recombination between the att sites (attR×attL and attB×attP) generates an expression vector and a by-product. The expression vector contains the nucleic acid sequence of interest recombined into the destination vector backbone. Following transformation and selection in E. coli, the expression vector is ready to be used for expression in the appropriate host.
The nucleic acid sequence of interest can be expressed from recombinant circular or linear DNA vector using any suitable promoter. Suitable promoters for expressing RNA from a vector include, e.g., the U6 or H1 RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art. The expression vector should have the necessary 5′ upstream and 3′ downstream regulatory elements such as promoter sequences, ribosome recognition and binding TATA box, and 3′ UTR AAUAAA (SEQ. ID. No. 25) transcription termination sequence for the efficient gene transcription and translation in its respective host cell. The recombinant vectors can also comprise inducible or regulatable promoters for expression of the nucleic acid sequence of interest in hematopoietic progenitor cells. The nucleic acids that are expressed from recombinant vectors can also be delivered to, and expressed directly in, cells. In one embodiment, the nucleic acids are expressed as RNA precursor molecules from a single vector, and the precursor molecules are processed into the functional miR gene product by a suitable processing system, including, but not limited to, processing systems extant within a cell. Other suitable processing systems include, e.g., the in vitro Drosophila cell lysate system (e.g., as described in U.S. Published Patent Application No. 2002/0086356 to Tuschl et al., the entire disclosure of which is incorporated herein by reference) and the E. coli RNAse III system (e.g., as described in U.S. Published Patent Application No. 2004/0014113 to Yang et al., the entire disclosure of which is incorporated herein by reference).
Selection of vectors suitable for expressing the nucleic acid sequence, methods for inserting nucleic acid sequences into vector to express the gene products, and methods of delivering the recombinant plasmid to the cells of interest are within the skill in the art. See, for example, Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al. (2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, the entire disclosures of which are incorporated herein by reference.
Examples of expression vectors for mammalian host cells include but are not limited to the strong CMV promoter-based pcDNA3.1 (INVITROGEN) and pCIneo vectors (Promega) for expression in mammalian cell lines such as CHO, COS, HEK-293, Jurkat, and MCF-7; replication incompetent adenoviral vector vectors pAdeno X, pAd5F35, pLP-Adeno-X-CMV (Clontech), pAd/CMV/V5-DEST, pAd-DEST vector (INVITROGEN) for adenovirus-mediated gene transfer and expression in mammalian cells; pLNCX2, pLXSN, and pLAPSN retrovirus vectors for use with the RETRO-XTM system from Clontech for retroviral-mediated gene transfer and expression in mammalian cells; pLENTI4/V5-DESTTM, pLenti6/V5-DESTTM, and pLENTI6.2/V5-GW/lacZ (INVITROGEN) for lentivirus-mediated gene transfer and expression in mammalian cells; adenovirus-associated virus expression vectors such as pAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC vector (Stratagene) for adeno-associated virus-mediated gene transfer and expression in mammalian cells;
A simplified system for generating recombinant adenoviruses is presented by He T C. et. al. Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998. The gene of interest is first cloned into a shuttle vector, e.g. pAdTrack-CMV. The resultant plasmid is linearized by digesting with restriction endonuclease Pme I, and subsequently cotransformed into E. coli. BJ5183 cells with an adenoviral backbone plasmid, e.g. pAdEasy-1 of Stratagene's AdEASYTM Adenoviral Vector System. Recombinant adenovirus vectors are selected for kanamycin resistance, and recombination confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmid is transfected into adenovirus packaging cell lines, for example HEK 293 cells (E1-transformed human embryonic kidney cells) or 911 (E1-transformed human embryonic retinal cells) (Human Gene Therapy 7:215-222, 1996). Recombinant adenovirus are generated within the HEK 293 cells.
In one embodiment, a recombinant lentivirus can be used for the delivery and expression of a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1, 3 or 4 in either dividing and non-dividing mammalian cells. The HIV-1 based lentivirus can effectively transduce a broader host range than the Moloney Leukemia Virus (MoMLV)-base retroviral systems. Preparation of the recombinant lentivirus can be achieved using the pLENTI4/V5-DESTTM, pLENTI6/V5-DESTTM or pLenti vectors together with ViraPowerTM Lentiviral Expression systems from Invitrogen.
In one embodiment, a recombinant adeno-associated virus (rAAV) vector can be used for the expression of a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1, 3 or 4. Because AAV is non-pathogenic and does not illicit an immune response, a multitude of pre-clinical studies have reported excellent safety profiles. rAAVs are capable of transducing a broad range of cell types and transduction is not dependent on active host cell division. High titers, >108 viral particle/ml, are easily obtained in the supernatant and 1011-1012 viral particle/ml with further concentration. The transgene is integrated into the host genome so expression is long term and stable.
The use of alternative AAV serotypes other than AAV-2 (Davidson et al (2000), PNAS 97(7)3428-32; Passini et al (2003), J. Virol 77(12):7034-40) has demonstrated different cell tropisms and increased transduction capabilities. With respect to brain cancers, the development of novel injection techniques into the brain, specifically convection enhanced delivery (CED; Bobo et al (1994), PNAS 91(6):2076-80; Nguyen et al (2001), Neuroreport 12(9):1961-4), has significantly enhanced the ability to transduce large areas of the brain with an AAV vector.
Large scale preparation of AAV vectors is made by a three-plasmid cotransfection of a packaging cell line: AAV vector carrying the chimeric DNA coding sequence, AAV RC vector containing AAV rep and cap genes, and adenovirus helper plasmid pDF6, into 50×150 mm plates of subconfluent 293 cells. Cells are harvested three days after transfection, and viruses are released by three freeze-thaw cycles or by sonication.
AAV vectors are then purified by two different methods depending on the serotype of the vector. AAV2 vector is purified by the single-step gravity-flow column purification method based on its affinity for heparin (Auricchio, A., et. al., 2001, Human Gene therapy 12; 71-6; Summerford, C. and R. Samulski, 1998, J. Virol. 72:1438-45; Summerford, C. and R. Samulski, 1999, Nat. Med. 5: 587-88). AAV2/1 and AAV2/5 vectors are currently purified by three sequential CsCl gradients. Delivery vectors can also included but are not limited to replication-defective adenoviral vectors, cationic liposomes and protein-cationic peptides. For example, one study reports a system to deliver DNA in vitro by covalently attaching the surfactant associated protein B (SP-B) to a 10 kDa poly-lysine. See, Baatz, J., et al., PNAS USA, 91:2547-2551 (1994). See, e.g., Longmuir, et al., 1992 ASBMB/Biophysical Society abstract; Longmuir, et al., 1993 Biophysical Society abstract.
Therapeutic Uses and Administration
In one embodiment, a nucleic acid or vector administered to the host cells comprise a non-cationic lipid for cytoplasmic and/or nuclear delivery, wherein the nucleic acid or vector is stable and is used in biological extracellular fluids typically found in animals, particularly blood serum.
Liposomes, spherical, self-enclosed vesicles composed of amphipathic lipids, have been widely studied and are employed as vectors for in vivo administration of therapeutic agents. In particular, the so-called long circulating liposomes formulations which avoid uptake by the organs of the mononuclear phagocyte system, primarily the liver and spleen, have found commercial applicability. Such long-circulating liposomes include a surface coat of flexible water soluble polymer chains, which act to prevent interaction between the liposome and the plasma components which play a role in liposome uptake. Alternatively, hyaluronan has been used as a surface coating to maintain long circulation.
In one embodiment, the liposome encapsulate the nucleic acid sequences, vectors or even the viral particles. In one embodiment, the nucleic acid sequences or vectors are condensed with a cationic polymer, e.g., PEI, polyamine spermidine, and spermine, or a cationic peptide, e.g., protamine and poly-lysine, and encapsulated in the lipid particle. The liposomes can comprise multiple layers assembled in a step-wise fashion.
Lipid materials well known and routinely utilized in the art to produce liposomes. Lipids may include relatively rigid varieties, such as sphingomyelin, or fluid types, such as phospholipids having unsaturated acyl chains. “Phospholipid” refers to any one phospholipid or combination of phospholipids capable of forming liposomes. Phosphatidylcholines (PC), including those obtained from egg, soy beans or other plant sources or those that are partially or wholly synthetic, or of variable lipid chain length and unsaturation are suitable for use in the present invention. Synthetic, semisynthetic and natural product phosphatidylcholines including, but not limited to, distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), soy phosphatidylcholine (soy PC), egg phosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine (HEPC), dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) are suitable phosphatidylcholines for use in this invention. All of these phospholipids are commercially available. Further, phosphatidylglycerols (PG) and phosphatic acid (PA) are also suitable phospholipids for use in the present invention and include, but are not limited to, dimyristoylphosphatidylglycerol (DMPG), dilaurylphosphatidylglycerol (DLPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG) dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dilaurylphosphatidic acid (DLPA), and dipalmitoylphosphatidic acid (DPPA). Distearoylphosphatidylglycerol (DSPG) is the preferred negatively charged lipid when used in formulations. Other suitable phospholipids include phosphatidylethanolamines, phosphatidylinositols, sphingomyelins, and phosphatidic acids containing lauric, myristic, stearoyl, and palmitic acid chains. For the purpose of stabilizing the lipid membrane, it is preferred to add an additional lipid component, such as cholesterol. Preferred lipids for producing liposomes according to the invention include phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in further combination with cholesterol (CH). According to one embodiment of the invention, a combination of lipids and cholesterol for producing the liposomes of the invention comprise a PE:PC:Chol molar ratio of 3:1:1. Further, incorporation of polyethylene glycol (PEG) containing phospholipids is also contemplated by the present invention.
In addition, in order to prevent the uptake of the liposomes into the cellular endothelial systems and enhance the uptake of the liposomes into the tissue of interest, the outer surface of the liposomes may be modified with a long-circulating agent. The modification of the liposomes with a hydrophilic polymer as the long-circulating agent is known to enable to prolong the half-life of the liposomes in the blood
Liposomes encapsulating the nucleic acid sequences described herein can be obtained by any method known to the skilled artisan. For example, the liposome preparation of the present invention can be produced by reverse phase evaporation (REV) method (see U.S. Pat. No. 4,235,871), infusion procedures, or detergent dilution. A review of these and other methods for producing liposomes may be found in the text Liposomes, Marc Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1. See also Szoka Jr. et al., (1980, Ann. Rev. Biophys. Bioeng., 9:467).
The use of an therapeutically effective amount of the nucleic acid sequences or vectors disclosed herein for the treatment of thrombocytopenia and anemia should preferably include but is not limited to a composition of the nucleic acid segments in lactated Ringer's solution and the composition is sterile. Lactated Ringer's solution is a solution that is isotonic with blood and intended for intravenous administration. Include are antioxidants, buffers, antibiotics and solutes that render the compositions substantially isotonic with the blood of an intended recipient. In another embodiment, the composition comprise gene delivery vectors described herein. In another embodiment, the composition also include water, polyols, glycerine and vegetable oils, and nutrients for cells, for example. Compositions adapted for parenteral administration can be presented in unit-dose or multi-dose containers, in a pharmaceutically acceptable dosage form. Such dosage forms, along with methods for their preparation, are known in the pharmaceutical and cosmetic art. Harry's Cosmeticology (Chemical Publishing, 7th ed. 1982); Remington's Pharmaceutical Sciences (Mack Publishing Co., 18th ed. 1990).
In one embodiment, dosage forms include pharmaceutically acceptable carriers that are inherently nontoxic and nontherapeutic. Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and polyethylene glycol.
In one embodiment, other ingredients can be added, including antioxidants, e.g., ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; and sugar alcohols such as mannitol or sorbitol.
In one embodiment, the administration of the nucleic acid segments or gene delivery vectors disclosed herein are by any suitable route, and means, for example, parenterally, intravenous, intra-arterial, intracranial, intracerebrospinal, intratumoral, peritoneal, by injection, by catheter, by implantation with or without a matrix or gel material, or by gradual delivery device. In one embodiment, the nucleic acid segments or gene delivery vectors described herein can be administered directly by injection.
The therapeutically effective amount amounts to be administered will depend on the severity of the condition and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art; however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reason.
This invention is further illustrated by the following example which should not be construed as limiting. The contents of all references cited throughout this application, as well as the figures and table are incorporated herein by reference.
EXAMPLES
Materials and Methods
MiRNA expression profiling and data analysis—miRNA expression profiling was performed using the plate capture method unless otherwise stated. 96-well PCR plates with N-oxysuccinimide surface (DNA-BIND plates, Corning Costar) were coated at room temperature for 1 hour with 5 μM mixture of 5′ amino-antisense oligonucleotides (see Table 4) at 20 μl per well according to manufacturer's protocol. Coated plates were successively washed with (100 mM Tris-HCl, pH8.0, 150 mM NaCl, 1 mM EDTA) and (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Total RNA (10 ng or higher) was diluted to 20 μl in 50 mM Tris-HCl, pH 8.0, 1 M NaCl, 1 mM EDTA, 1×RNAsecure (Ambion), containing pre-control synthetic miRNA mixture at the ratio previously described (1) miRNAs were captured in the coated wells by denaturing at 80° C. for 5 minutes and gradually cooling to room temperature in 1.5 hours in a PCR machine, followed by three 2×SSC washes. 3′ adaptor ligation and 5′ adaptor ligations were carried out as described (1) in 20 μl reaction volumes, with four 2×SSC washes after each ligation. Ligated miRNA were denatured for 5 minutes at 80° C. in 20 μl water with 2 μM adaptor-specific RT primer, chilled on ice, and reverse transcribed as described (1). RT products were denatured at 95° C. for 5 minutes before 2 rounds of PCR amplification with described conditions (1), for 26 and 27 cycles respectively, to incorporate biotin labels for low input RNA profiling. Labeled miRNAs were hybridized to a bead-based detection platform (1), with updated detection probes (Table 5). Median fluorescence intensities were quantitated on a Luminex 100S machine (Luminex Corp).
Data were normalized as described (1) with modifications. Average readings from 5 water-only labeled samples were used for probe-specific background subtraction.
Linear normalization among different bead sets for the same sample was performed using readings from 2 post-control probes with equal contribution. Sample normalization was subsequently carried out assuming equal total fluorescence readings. To identify markers, all ERY samples were compared to all MEGA samples, with median-based t-test and 50,000 permutations, using the Comparative Marker Selection module in GENEPATTERN (2).
Cell sorting and flow cytometry—Human umbilical cord blood was harvested at Brigham and Women's Hospital with informed patient consent under an IRB approved protocol. Adult human bone marrow cells were obtained from AllCells, LLC. Mononuclear cells were purified by Ficoll Hypaque sedimentation. Lineage depletion was performed using antibodies against CD2, CD3, CD4, CD5, CD8, CD11b, CD14C, CD19, and CD56 with a magnetic column (Miltenyi Biotec). Populations were defined as follows: MEP (CD34+CD38iL3Ra−CD45RA−), ERY1 (CD34+CD71+GlyA−), ERY2 (CD34−CD71+GlyA−), ERY3 (CD34−CD71+GlyA+). Megakaryocytes were purified without lineage depletion according to the following immunophenotypes: MEGA1 (CD34+CD61+CD41+CD45−), MEGA2 (CD34−CD61+CD41+CD45−). Adult human bone marrow cells were similarly sorted according to CD34−CD61+CD41+ and CD34−CD71+GlyA+. Sorting was performed with a Vantage SE Diva or with an Aria (BD Biosciences). RNA was extracted using TRIZOL (INVITROGEN).
For the in vitro human primary culture experiment, approximately 500,000 cells were stained with CD41-FITC, Ter119-PE and CD71-PE-Cy5 antibodies for 15 minutes on ice and washed twice before flow cytometry. For analysis of transplant recipients, murine bone marrow cells were labeled with CD41-PE and Ter119-APC, or Ter119-APC and CD71-PE. Peripheral blood cells were harvested into 0.3 8% sodium citrate and stained with CD41-PE.
Mouse MEPs were purified as described in (3). Briefly, bone marrow cells were harvested from 8- to 10-week old C57Bl6/J mice and stained with an antibody cocktail containing biotinylated lineage markers including Ter119, CD3, CD4, CD8, CD11b/Mac-1 Gr-1 and B220, and followed by staining with a second antibody cocktail containing streptavidin-PerCP, Sca-PE, cKit-APC, CD16/32 PE-Cy7 and CD34-FITC. MEPs are defined as the Lin-cKit+Sca-CD34-CD16/32-population. Antibodies used for cell surface markers are found in Table 2.
Constructs—Expression vectors for hsa-miR-150 contain a 473 bp genomic fragment that includes the hairpin region of hsa-miR-150 and  ̃200 bp of flanking sequence on each side. This genomic expression cassette was PCR amplified from human genomic DNA (Roche Applied Science) with primers containing 5′ linker sequences harboring relevant digestion sites (core primer sequences: 5′ CAGCATAGGGTGGAGTGGGT3′ (SEQ. ID. No. 5); 5′TACTTTGCGCATCACACAGA3′ (SEQ. ID. No. 6)). For the human CD34+ primary culture experiment, the lenti-viral vector pLKO. 1 (obtained from The RNAi Consortium, Broad Institute) was used, with the miR-150 expression cassette, or an shRNA against luciferase (shLuc), cloned into the AgeI and EcoRI sites. hsa-miR-15b-16-2 was similarly cloned with a genomic DNA fragment through PCR amplification (core primer sequences: 5′TTTCCTCAAAACAGGAAGG3′ (SEQ. ID. No. 7); 5′CCACCAAGTAAGTCATTTTC3′ (SEQ. ID. No. 8)). For expression in cell lines, the miR-150 expression cassette, or EGFP coding sequence, was cloned into the pMSCV-puro vector through the BglII and MluI sites. For in vivo transplantation assays, the pMSCV-puro vector was substituted with pMSCV-EGFP, in which the EGFP coding sequence replaced that of the puromycin resistance gene in pMSCV-puro.
Mutant miR-150 constructs were created by PCR-mediated site-directed mutagenesis. Mutations were introduced into the 5′ seed region of mature hsa-miR-150, as well as into the opposite arm of the hairpin to maintain overall hairpin structure. Primers used are listed below:









(SEQ. ID. No. 9)



5′CCCCATGGCCCTGTCTGGGAACCCTTGTACCAGTG3′



 



(SEQ. ID. No. 10)



5′ CACTGGTACAAGGGTTCCCAGACAGGGCCATGGGG3′



 



(SEQ. ID. No. 11)



5′ CCCTGGTACAGGCCTCCCGGACAGGGACCTG3′



 



(SEQ. ID. No. 12)



5′ CAGGTCCCTGTCCGGGAGGCCTGTACCAGGG3′.






The MYB cDNA clone, containing only the coding sequence and Kozak sequence, was obtained from Invitrogen (Ultimate ORF collection) in the form of a Gateway entry vector. This clone, as well as a Gateway entry clone without insert (vector control), were recombined into pLenti6.2/V5DEST vector using LR recombination reactions (Invitrogen).
MYB 3′UTR luciferase reporter was created by inserting human MYB 3′UTR (according to RefSeq NM—005375) into the XhoI and NotI sites in the psiCHECK2 vector (Promega), downstream of the renilla luciferase coding sequence. MYB 3′UTR was amplified from human genomic DNA with the following primers:









(SEQ. ID. No. 13)



5′TAACTCGAGACATTTCCAGAAAAGCATTATG3′,



and



 



(SEQ. ID. No. 14)



5′ATAGCGGCCGCAGGTAAAATAAGGGCACATC3′.






Mutations of putative miR-150 binding sites were created by PCR-mediated site-directed mutagenesis. Primers used are listed below.









Site 1:



(SEQ. ID. No. 15)



5′ACTTTTCATGAATCCCAGAAGAACCTAT3′



 



(SEQ. ID. No. 16)



5′ATAGGTTCTTCTGGGATTCATGAAAAGT3′



 



Site 2:



(SEQ. ID. No. 17)



5′TGAAAACTTGTTTCCCAGACTCTGCATT3′



 



(SEQ. ID. No. 18)



5′AATGCAGAGTCTGGGAAACAAGTTTTCA3′



 



Site 3:



(SEQ. ID. No. 19)



5′TGCACTTCTTTTTTCCCAGATGTGTGTTGT3′



 



(SEQ. ID. No. 20)



5′ACAACACACAT CT GGGAAAAAAGAAGT GCA3′



 



Site 4:



(SEQ. ID. No. 21)



5′CTGTTTTATAATTTCCCAGTTCTGCATTTG3′



 



(SEQ. ID. No. 22)



5′ CAAAT GCAGAAC T GGGAAAT TATAAAACAG3′






Short hairpin RNAs against human MYB were obtained from The RNAi Consortium (world wide web “period” broad “period” mit “period” edu “forward slash” genome “underscore” bio “forward slash” trc “forward slash”). The IDs of the shMYB-1 and shMYB-2 clones are TRCN0000040058 and TRCN0000009853.
Quantitative RT-PCR—Quantitative RT-PCR primers and probes were all obtained from Applied Biosystems. Reverse transcription reactions were performed following the manufacturer's protocol with minor modifications. Briefly, 1 ng to 10 ng of total RNA were reverse transcribed using the MultiScribe cDNA synthesis system (Applied Biosystems) in 5 μl volume with either miRNA gene specific RT primers, or with 6.25 ng random primers (Invitrogen). Duplicate or triplicate RT reactions were performed for each sample and each RT primer. RT products were diluted 2.5 fold before PCR. PCR reactions were performed in duplicate for each RT product, following the manufacturer's protocol and using assays from Applied Biosystems on an ABI HT7900 real time PCR machine. Reactions for eukaryotic 18S ribosomal RNA and messenger RNAs were performed with random-primer-based RT products, whereas reactions for miRNAs used corresponding gene-specific RT products. Threshold cycles (using a manual cutoff of 0.2) or genes of interest were normalized by Ct values of corresponding 18S rRNA reactions. ΔCt values (Ct of 18S minus Ct of gene of interest) were used unless specified otherwise. Quantitative RT-PCR assays used in this study are found in Table 1.
In vitro primary culture of human CD34+ cells-Cryopreserved human adult bone marrow CD34+ cells were obtained from Cambrex (Poietics; Cambrex). Cells were cultured in Serum Free Expansion Medium (SFEM, Stem Cell Technologies) supplemented with 100 U/mL penicillin/streptomycin, 2 mM glutamine, and 40 μg/mL lipids (SIGMA ALDRICH). Erythroid and megakaryocytic differentiation were supported in a single liquid culture, similarly as described (4), in the presence of 50 ng/mL TPO, 100 ng/mL SCF, 10 ng/mL IL-3, 10 ng/mL IL-6, and 0.5 U/mL EPO. The concentration of EPO was increased to 3 IU/mL on day 7. Cells were harvested for flow cytometry following 10 days of liquid culture. Lentiviral infection was performed starting one day after thawing cells. Where indicated, cDNA construct and miRNA construct were infected on consecutive days. Cells were selected with 2 μg/mL puromycin or 3 μg/mL blasticidin one day after infection.
Murine bone marrow transplant—All mice were purchased from the Jackson Laboratory. Murine bone marrow transplant was performed similarly as previously described (5), and approved by the MGH Subcommittee on Research Animal Care. Donor C57Bl6/J mice ( ̃8 weeks) were primed with 150 mg/kg 5FU for four days. Bone marrow cells were purified by Ficoll (GE Healthcare) density gradient centrifugation, following the manufacturer's protocol. Cells were transduced with empty vector or miR-150 retrovirus in X-VIVO 15 medium (Biowhittaker) supplemented with 100 ng/mL SCF, 50 ng/mL TPO, 50 ng/mL Flt3 ligand and 20 ng/mL IL3 by centrifugation onto plates coated with retronectin (TAKARA). Lethally irradiated (9.5 Gy) recipient mice were transplanted with 2.5-4 million cells the day after infection. Hematopoeitic recovery was monitored by complete blood count. Bone marrow cells of 7 pairs of recipients were analyzed at 5 to 8 weeks post-transplantation respectively. Platelets were analyzed 7-weeks post-transplantation.
Cell culture—K562 and 293T cells were obtained from ATCC, and were cultured according to ATCC instructions.
Mouse bone marrow cells were treated with antagomir (50 μg/ml) or PBS for three days in X-VIVO 15 medium (Biowhittaker) supplemented with 50 ng/mL SCF, 50 ng/mL TPO, 50 ng/mL Flt3 ligand and 20 ng/mL IL3. Cells were then harvested for RNA analysis.
Oligonucleotides and antagomirs-DNA oligonucleotides were synthesized by IDT Technology. RNA oligonucleotides, including antagomirs and DNA-RNA hybrids, were synthesized by Dharmacon. Antagomir stock solution was prepared in PBS.







Antagomir-150:


(SEQ. ID. No. 23)


5′


 


mC(*)mA(*)mCmUmGmGmUmAmCmAmAmGmGmGmUmUmGmGmG


 


(*)mA(*)mG


 


(*)mA (*)(3′-Chl) 3′.


 


Antagomir-scrambled:


(SEQ. ID. No. 24)


5′mC(*)mU(*)mCmGmCmGmUmAmGmA


 


mAmGmAmGmUmAmGmGmU(*)mG(*)mG(*)mA(*) (3′-Chl) 3′.


 


(mN: 2′OMe base; *: phosphorothioate linkage;


 


Chl: cholesterol).






Colony assay—Megakaryocyte colony assay was performed using the MegaCult-C kit (Stem Cell Technology) according to the manufacturer's protocol. Bone marrow cells from recipient mice 7 to 10 weeks after transplantation were sorted into GFP− and GFP+ populations. Two recipient mice were analyzed for each construct, and each population of cells was assayed in duplicates with 100,000 sorted bone marrow cells per well. Cultures were maintained for 8 days before stained for acetylcholinesterase activity and scored. For antagomir treatment, 1000 LKS cells or 4000 MEPs were FACS-sorted and assayed in the presence of antagomir (50 μg/ml) or PBS and maintained in culture for 11 days before staining and scoring. In all cases, a colony with ≧3 acetylcholinesterase-positive cells was scored as a megakaryocyte colony.
For erythoid colony assay, 5FU primed wild type C57Bl6/J marrow was transduced as described above. Forty-eight hours after viral transduction, 30,000 GFP+ cells were FACS-sorted and plated into methylcellulose (StemCell Technologies, M3334) which only contains EPO.
Anemic response—Phenylhydrazine hydrochloride (Sigma) solution in PBS was injected intraperitoneally into 10- to 12-week wild type C57Bl6/J mouse (60 mg/kg body weight) on each of days 0, 1, and 2. On the 3rd day, mice were euthanized by CO2 inhalation and bone marrow was harvested and stained with a lineage marker antibody cocktail as described in cell sorting and flow cytometry. Lineage negative cells were FACS sorted into TRIZOL reagent for RNA preparation.
Western blot analysis—Western blot analysis was performed as previously described (6). MYB antibody (clone 1-1) was from Upstate Biotechnology. Beta tubulin antibody (ab6046) was from Abcam.
Luciferase reporter assay—293T cells were plated in 96 well plates at 5000 cells per well the day before transfection. Transfection was carried out in 8 replicates using FuGENE 6 (Roche), with 100 ng of plasmid mixture (90 ng of expression vector and 10 ng of reporter vector in the psiCHECK2 backbone). Luciferase assays for both firefly and renilla luciferase were performed 2 days after transfection, using the Dual-Glo Luciferase assay kit (Promega). Luminescence was quantitated on a Tecan Spectrafluor Plus machine. Renilla luciferase readings were normalized against the firefly luciferase activity in the corresponding well.
Statistical Analysis—Student's t-test (2 tailed, unequal variance) was used for statistical analysis on experiments, unless otherwise specified.
Example 1
miRNA Expression in Hematopoietic Progenitor Cells
Mammalian developmental cell fate can be guided at least in part by different mechanism of gene regulation, such as by miRNAs. These recently discovered  ̃22 nt non-coding RNAs negatively regulate the expression of target proteins either by inhibiting translation of their cognate mRNAs, or by inducing mRNA degradation, primarily through sites in the 3′UTR (see review (6)). Previous miRNA expression patterns encode developmental history supports a role of miRNAs in lineage specification (7). Here, MEP differentiation was used as a model system to test that miRNA can regulate cell fate, starting with the profiling of the expression of miRNAs in MEPs, erythroid and megakaryocytic primary cells.
Unfortunately, the small number of precursor cells obtainable from human donors has precluded a thorough analysis of miRNA expression in hematopoiesis (and other systems) using conventional miRNA profiling methods, which generally require either large amounts of input RNA, or are not amenable to genome-wide, high throughput applications. To address this technical challenge, a method was developed in which mature miRNAs are captured in 96-well plates using immobilized 5′-amino-modified oligonucleotides complementary to the mature miRNA sequence of more than 300 human miRNAs (from the miRBASE (8) release 7.0). This plate-based capture obviates the need for gel-purification of small RNAs which, in addition to being labor-intensive, results in significant loss of input miRNAs. The captured miRNAs were ligated with adaptors on 3′ and 5′ ends successively, reverse transcribed, and amplified via PCR (FIG. 1A). The biotinylated PCR products were then detected by hybridization to fluorescent beads coupled to capture oligonucleotides complementary to the mature miRNA sequence. The beads were then analyzed by flow cytometry, where the color of the bead indicates the identity of the miRNA and the phycoerythrin channel indicates the abundance of that particular miRNA (7). In contrast to previously reported method (7) that required  ̃10 μg of input RNA, the present method yields similarly informative results with as little as 10 ng of total RNA (FIG. 5). FIG. 5A shows the reproducibility performance of the plate capture method of miRNA labeling. Two experiments of miRNA profiling with the plate capture method were performed on total RNA from MCF-7, 293T or K562 cells. Data were normalized and log 2-transformed. Data from experiment 1 were plotted against data from experiment 2. Each dot represents the reading of one miRNA in one sample. FIG. 5B shows data that were normalized and log 2-transformed. The differences of log 2-transformed data between MCF-7 and 293T cells, which reflect the fold change, were plotted to compare the two labeling methods. Each dot represents one miRNA. Note that most dots are close to the diagonal, indicating the two labeling methods captured similar results.
With a suitable miRNA profiling method in hand, the miRNA expression pattern in MEPs and early megakaryocytic and erythroid populations obtained from FACS-sorted human umbilical cord blood samples were examined. Using well-established surface markers, 6 populations of cells were purified, and them are referred to as MEP (CD34+,CD38+,IL-3Rα−, CD45RA−), MEGA1 (CD34+,CD41+,CD61+,CD45−), MEGA2 (CD34−,CD41+,CD61+,CD45−), ERY1 (CD34+,CD71+,GlyA−), ERY2 (CD34−,CD71+,GlyA−) and ERY3 (CD34−, CD71+,GlyA+). These 6 populations thus capture the bifurcation of the megakaryocytic and erythroid lineages with fine granularity (FIG. 1C).
Profiling of 320 miRNAs was performed to identify those most differentially expressed across the megakaryocytic and erythroid populations. The profiling result (FIG. 1B, Table 6) captured and extended known miRNA expression patterns. For example, miR-451 and miR-144 were highly expressed in CD71+GlyA+ erythrocytes, and miR-222 was down-regulated during erythropoiesis (9, 10) (FIG. 1B). Among all miRNAs, differential analysis (Table 3) identified miR-150 with the most divergent expression between early megakaryocytic and erythroid cells, being expressed >15-fold higher in megakaryocytes (FIG. 1C). Quantitative RT-PCR analysis in sorted umbilical cord blood cells confirmed miR-150 as being highly expressed in megakaryocytes, weakly expressed in erythrocytes and moderately expressed in MEPs (FIG. 1D, FIG. 16). Similarly, miR-150 was expressed more highly in CD41+CD61+ megakaryocytes than in CD71+ GlyA+ erythrocytes in human adult bone marrow (FIG. 6B).
Example 2
Function of miR-150 in Differentiation of Progenitor Cells
While high level expression of miR-150 was observed in the megakaryocytic lineage, it is conceivable that it may not play a functional role in the specification of megakaryocytes versus erythrocytes. Arguing for its functional importance is its exquisite sequence conservation across organisms with functional erythrocytic and thrombocytic systems, exhibiting identical sequence in the 5′ seed region that mediates target recognition (FIG. 7). To assess a potential causal role of miR-150 in the specification of megakaryocytes versus erythrocytes, a series of gain- and loss-of-function experiments were performed.
First, a bi-lineage primary cell culture was used, in which human CD34+ hematopoietic progenitor cells isolated from adult human bone marrow, when cultured in the presence of thrombopoietin and erythropoietin, differentiate along the megakaryocytic and erythroid lineages in vitro. This system allowed the quantitative perturbation in the balance of megakaryocytic and erythroid development from progenitor cells. CD34+ cells were transfected with a lentiviral construct harboring miR-150, resulting in a physiological level of miR-150 expression that is similar to that observed in primary megakaryocytes (FIG. 8).
The function of miR-150 was assayed in an in vitro primary culture. CD34+ hematopoietic progenitors derived from human adult bone marrow cells were transduced with constructs expressing a control hairpin (shLuc), miR-150, a mutant miR-150 or miR-15b-16-2. The culture was analyzed after 10 days of differentiation, using flow cytometry with lineage markers CD41 (megakaryocytic) and GlyA (erythroid). Transduced cells were allowed to differentiate. Megakaryocytes were then enumerated by flow cytometry measuring the CD41+GlyA− population. Compared to CD34+ cells transduced with a control vector (shLuc, expressing a short hairpin RNA against luciferase), mutant miR-150, or an irrelevant miRNA construct (miR-15b-16-2), the miR-150 expressing cells yielded an average of 8-fold enrichment of megakaryocytes (FIG. 2A, 2B). This result indicates that miR-150 shifts the balance of megakaryocytic-erythroid differentiation towards megakaryocytes, and further indicates its role in governing the fate of MEPs.
Example 3
Function of miR-150 in In Vivo Differentiation of Progenitor Cells
Having established a functionally important role of miR-150 in a human in vitro model of MEP differentiation, the functions of miR-150 in vivo were examined to address whether miR-150 inhibits the erythroid lineage, promotes the megakaryocytic lineage, or both. To this end, a murine bone marrow transplantation model was used, in which stem/progenitor-cell-enriched bone marrow cells from donor mice were transduced with either miR-150 retrovirus or control virus at low titer. The vectors carry a GFP marker, thus labeling transduced cells and cells derived from them with green fluorescence. The mixture of transduced and non-transduced donor cells was transplanted into lethally irradiated recipients. Bone marrow and peripheral blood of recipients were analyzed 5 to 8 weeks post transplantation, when the hematopoietic system had largely recovered in the hosts. Both viral vectors carry GFP as a marker, allowed the distinguishing between donor-derived cells that were transduced from those that were not (FIG. 9).
miR-150 was expressed from a retroviral vector with a GFP marker. This construct, or a control vector, was assayed by murine bone marrow transplant. Recipient mice were analyzed 5 to 8 weeks post-transplantation on transduced (GFP+) and non-transduced (GFP−) cells. Flow cytometry was used to assay the bone marrow cells with megakaryocyte-(CD41) and erythrocyte-(Ter119) specific markers. Strikingly, compared to either non-transduced (GFP−) cells in miR-150 recipients, or vector control recipient mice, miR-150 transduced (GFP+) bone marrow cells exhibited a dramatic (>15-fold on average) expansion of megakaryocytes (CD41+Ter119−) in relation to all transduced cells in the bone marrow (FIG. 2C, 2E, 10). Recipient bone marrow cells were FACS sorted into GFP+ and GFP− populations, and measured for PF4 expression using quantitative RT-PCR. Each pair of bars represents data from one recipient mouse. It was observed that a strong elevation in the expression of the megakaryocyte-specific gene PF4 (11) occurred in miR-150 transduced bone marrow cells (FIG. 2F). In addition, the circulating platelets were analyzed in the peripheral blood of recipient animals 7-week post-transplantation. The ratio of GFP+ platelet percentage to the percentage of GFP+ bone marrow cells was plotted to reflect the thrombocytogenic potential of bone marrow cells. n=5. There was a consistent 2- to 14-fold enrichment in GFP+ circulating platelets in miR-150 animals compared to control vector recipients (FIG. 2G, 11). These results proved that miR-150 expression led to a bona fide increase in bone marrow megakaryocytes that were competent to produce mature platelets in circulation.
In contrast, an over 60% decrease in GFP+ erythrocytes (Ter119+CD41−) was observed, again in relation to all GFP+ cells in the bone marrow (FIG. 2D, 2E). Importantly, the absolute numbers of GFP+ megakaryocytes in the bone marrow increased, whereas that of erythrocytes decreased in the presence of miR-150 expression (FIG. 12). Bone marrow cells with CD71 and Ter119 were examined, which distinguish different stages of murine erythroid differentiation (12). The R1 to R4 gates on CD71/Ter119 plot show that miR-150 expressing cells displayed a strong shift toward an earlier (immature) erythroid state, compared to controls (FIG. 2H, 2I, 2J). The percentage of R1 population among all erythrocytes (sum of R1 to R4) was used to derive a ratio between GFP+ and GFP− population within the same recipient mouse. n=7. P<0.002. Specifically, miR-150 expression led to an average of 8-fold increase in the immature R1 population, and a more than 60% decrease in the late R4 stage. These experiments indicate that ectopic miR-150 expression induces a blockage in the earliest definable stage of murine adult erythropoiesis, in addition to causing a significant reduction in the total erythroid population.
The megakaryocyte-promoting effect of miR-150 could be due to its effect on MEP commitment, or simply an effect on post-commitment megakaryocytic proliferation or survival. To address this, colony formation assay was used to quantify the megakaryocytic potential of progenitor cells at the single cell level. Erythroid colony formation assays were performed with 30,000 transduced bone marrow cells. Bone marrow cells from 5FU treated mice were transduced with a control vector or miR-150. GFP+ cells were sorted two days after transduction and assayed for erythroid colony forming units (CFU-E). Using the bone marrow cells from the transplant recipients, it was found that miR-150 overexpression resulted in a statistically significant increase in megakaryocyte colony-forming units (CFU-Mk) (FIG. 3A), coupled with a dramatic decrease in erythroid colony formation (FIG. 13). Representative erythroid colonies from control vector- or miR-150-transduced bone marrow cells. Note that rare erythroid colonies formed from miR-150 transduced cells showed reduced cell number. These gain of function experiments indicate that miR-150 regulates MEP fate, and not simply post-commitment megakaryocyte expansion.
Example 4
Effects of Loss of Function of miR-150 in In Vitro Differentiation of Progenitor Cells
To complement these miR-150 forced expression studies, a loss-of-function approach was used. MEP cells were isolated from bone marrow, and assayed for megakaryocyte colony formation in the presence or absence of an antagomir (a cholesterol-modified antisense oligonucleotide (13) directed against miR-150. Murine bone marrow cells were cultured in the presence of solvent (PBS), antagomir against miR-150 (anti-150) or a scrambled antagomir. miR-150 expression was measured with quantitative RT-PCR after 3 days of treatment (FIG. 14). Data represent 2ΔCt. Error bars represent standard deviation of measurement. MEPs treated with antagomir-150 showed more than a 4-fold decrease in CFU-Mk, compared to controls (see FIG. 3B). A similar effect was observed using purified uncommitted Lin-Kit+Sca+ hematopoietic stem cells (FIG. 3C). Interestingly, miR-150 knock-down megakaryocyte colonies showed normal morphology and acetylcholinesterase activity (FIG. 15), indicating that miR-150 might be dispensable once MEP commitment to the megakaryocyte lineage is established. To confirm a role of miR-150 in the physiological regulation of MEP fate, we treated mice with the anemia-inducing drug phenylhydrazine, and then measured miR-150 expression in lineage-negative bone marrow cells. miR-150 expression was significantly decreased in this setting of increased demand for erythropoiesis, consistent with miR-150's role in promoting megakaryopoiesis at the expense of erythropoiesis (FIG. 3D).
Example 5
miR-150 Regulates the c-myc Gene
The experiments described above firmly establish an important role of miR-150 in the specification of megakaryocytes from MEPs. To determine the mRNA targets of miR-150 that explain its effect on megakaryocytic/erythroid outcome, the targets predicted in common among several sequence-based prediction algorithms (14-16) were analyzed. MYB (also known as c-myb) was tested as a candidate because several recently reported mouse models, in which MYB activity was reduced due to either mutation or the serendipitous integration of a transgene near the MYB locus, displayed thrombocytosis and anemia (17-21). The expression of MYB messenger RNA, however, is not immediately indicative of a role in MEP differentiation, as similar expression in MEPs and early erythroid and megakaryocyte populations were noted (FIG. 16). Examination of the human MYB 3′UTR, however, identified multiple conserved miR-150 putative sites (FIG. 17). To establish miR-150 as a functional negative regulator of MYB, the erythroblastic cell line K562 was experimented in, which has the potential to be induced to differentiate into erythroid or megakaryocytic cells. A dramatic reduction in MYB protein level upon ectopic expression of miR-150 (FIG. 4A) was observed. Next, the MYB 3′ UTR was cloned into a luciferase reporter, and it was found that miR-150 repressed reporter activity by more than 6-fold, again consistent with miR-150 targeting MYB. Importantly, mutation of the 4 candidate miR-150 binding sites abrogated miR-150 repression, and a mutant miR-150 construct designed to be complementary to the mutant MYB 3′UTR binding sites restored miR-150 mediated repression, but did not affect the wild-type MYB 3′UTR (FIG. 4B, 4C). These experiments establish that miR-150 negatively regulates the protein level of MYB directly through its 3′ UTR.
Lastly, the question of whether miR-150 repression of MYB explains miR-150's erythroid/megakaryocytic effects was addressed. Using the in vitro CD34+ human bone marrow cell culture, consistent with reports that mice with reduced MYB activity display megakaryocytosis (17-21), two independent shRNA constructs that knocked down MYB expression (FIG. 18A) promoted megakaryocyte development (FIG. 18B). In contrast, forced expression of a MYB cDNA construct lacking its 3′UTR resulted in a decrease in megakaryocytes (FIG. 4D, 4E). Moreover, the MYB expression construct rescued the megakaryocytopoiesis-promoting effect of miR-150 to a large extent, indicating that MYB is a functionally relevant downstream effector of miR-150 (FIG. 4D, 4E). This MYB effect is consistent with a recent study showing MEPs with reduced MYB activity favor decision towards megakaryocytic as opposed to erythroid differentiation (20). In contrast to total loss of MYB function, which results in complete hematopoietic failure (19), our data indicate that modest modulation of MYB expression levels by miRNA can have important effects on lineage specification. These results are particularly interesting in light of the recent report of miR-150 regulation of MYB activity in B-lineage lymphocytes (22). It has been generally assumed that miRNAs have a plethora of targets that vary depending on cellular context. Remarkably, MYB appears to be a critical target of miR-150 both in establishing MEP fate and in regulating the differentiation of lineage-committed B-cells. This observation indicates that a single mRNA target of miRNAs might explain much of the miRNAs function, even in completely different developmental contexts. Our studies, of course, do not exclude the possibility that additional factors may also contribute to the establishment of MEP fate.
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TABLE 1







Quantitative RT-PCR assays used in this study










Gene of interest
Assay







Human Myb
Hs00193527_m1



Eukaryotic 18S
4333760T



miR-150
4373127



Mouse PF4
Mm00451315_g1

















TABLE 2







Antibodies for cell surface markers










Antigen
Fluorophore
Clone
Source





Mouse CD71
PE
C2
BD Pharmingen


Mouse Ter119
APC
TER-1 19
BD Pharmingen


Mouse CD41
PE
MWReg30
BD Pharmingen


Mouse CD3ε
biotin
145-2C1 1
BD Pharmingen


Mouse CD4
biotin
L3T4
BD Pharmingen


Mouse CD8a
biotin
53-6.7
BD Pharmingen


Mouse
biotin
RA3-6B2
BD Pharmingen


CD45R/B220


Mouse Ter119
biotin
TER-1 19
BD Pharmingen


Mouse CD1 1b
biotin
M1/70
BD Pharmingen


Mouse Gr1
biotin
RB6-8C5
BD Pharmingen


Mouse c-Kit
APC
2B8
BD Pharmingen


Mouse Sca-1
PE
Cat# MSCA04-3
Caltag


Mouse CD34
Pacific
RAM34
eBioscience



blue


Mouse
PE-Cy7
93
eBioscience


CD16/32


Streptavidine
PerCP
Cat# 554064
BD Pharmingen


Human CD71
FITC and
C2
BD Pharmingen



PE-Cy5


Human CD34
APC
581
BD Pharmingen


Humn CD61
FITC
2C9.G2
BD Pharmingen


Human CD41
PE and
HIP8
BD Pharmingen



FITC


Human CD45
APC-Cy7
2D1
BD Pharmingen


Human CD45RA
FITC
L48
BD Pharmingen


Human GlyA
PE
CLB-ery-1
CALTAG/




(AME-1)
Invitrogen
















TABLE 3







Comparative marker selection result for ERY samples vs. MEGA samples













Feature
Description
TTEST_Score
Feature P
FDR(BH)
Q Value
FWER
















EAM217
hmr-miR-
−6.134193365
6.00E−04
0.00373
0.00197
0.00148



150_rfam7.0







EAM161
hmr-miR-
−4.939403406
7.60E−04
0.00441
0.00225
0.02214



28_rfam7.0







EAM
hmr-miR-142-
−3.738371725
0.0012
0.00653
0.00333
0.26634


163
3p_rfam7.0







EAM371
hmr-miR-
−3.42554054
6.00E−04
0.00373
0.00197
0.45006



342_rfam7.0







EAM278
hmr-miR-98 rfam7.0
−2.987417405
0.00524
0.02682
0.01368
0.76548


EAM263
hmr-miR-
−2.93748816
0
0
0
0.79334



26a_rfam7.0







EAM224
hmr-miR-17-
2.052993319
0.00604
0.02919
0.01489
0.99828



5p_rfam7.0









Normalized miRNA expression data for ERY populations (ERY1, ERY2, ERY3) and MEGA populations (MEGA1, MEGA2) were log 2 transformed, thresholded at 6, and filtered to retain miRNAs with maximum expression over 8. Markers were selected using the ComparativeMarkerSelection module in GenePattern, with median-based t-test and 50,000 permutations. The table below shows features with BH-FDR of less than 0.05. Negative TTEST_Score means higher expression in MEGA samples, whereas positive number reflects higher expression in ERY samples. The table was sorted according to TTEST_Score. Feature: miRNA detection probe ID; Description: Detection probe annotation based on miRBASE 7.0; TTEST_Score: Median-based t-test score; Feature P: Nominal P value, after 50,000 permutations; FDR(BH): Benjamini-Hochberg false discovery rate; Q Value: q-value; FWER: Family-wise error rate.







TABLE 4





List of capture probes for initial miRNA capture


/5AmMC6/ indicates 5′ amino modification. Probes were 


synthesized by IDT.
















/5AmMC6/ACTCAGAAGGACAAGTAGAGTTTT
/5AmMC6/GTGGTAATCCCTGGCAATGTGAT


(SEQ. ID. No. 26)
(SEQ. ID. No. 27)


 


/5AmMC6/ACACTCTAAAGGGAACCATTTT
/5AmMC6/GGAAATCCCTGGCAATGTGAT


(SEQ. ID. No. 28)
(SEQ. ID. No. 29)


 


/5AmMC6/AAAGAAGTGCACCATGTTTGTTT
/5AmMC6/AAAGTGTCAGATACGGTGTGG


(SEQ. ID. No. 30)
(SEQ. ID. No. 31)


 


/5AmMC6/AAGAAGTGCACCGCGAATGT
/5AmMC6/ACAGTTCTTCAACTGGCAGCTT


(SEQ. ID. No. 32)
(SEQ. ID. No. 33)


 


/5AmMC6/AACACTCTGAAGGGAAGCGC
/5AmMC6/CTACCTGCACTATAAGCACTTTA


(SEQ. ID. No. 34)
(SEQ. ID. No. 35)


 


/5AmMC6/CACTCTAAAAGGATGCACTTT
/5AmMC6/TCAGTTTTGCATGGATTTGCACA


(SEQ. ID. No. 36)
(SEQ. ID. No. 37)


 


/5AmMC6/TTCACCAAAGGGAAGCACTTT
/5AmMC6/CCCAACAACATGAAACTACCTA


(SEQ. ID. No. 38)
(SEQ. ID. No. 39)


 


/5AmMC6/ACACTCTAAAGGGAAGTGCGTT
/5AmMC6/ACAAAGTTCTGTAGTGCACTGA


(SEQ. ID. No. 40)
(SEQ. ID. No. 41)


 


/5AmMC6/CTCCCTTCTTTCCTCCCGTC
/5AmMC6/CTAGTACATCATCTATACTGTA


(SEQ. ID. No. 42)
(SEQ. ID. No. 43)


 


/5AmMC6/CTCACACCTAGGTTCCAAGGATT
/5AmMC6/tgAGCTACAGTGCTTCATCTCA


(SEQ. ID. No. 44)
(SEQ. ID. No. 45)


 


/5AmMC6/TTACAGATGGATACCGTGCAATT
/5AmMC6/CCATCTTTACCAGACAGTGTT


(SEQ. ID. No. 46)
(SEQ. ID. No. 47)


 


/5AmMC6/CCACGACCGACGCCACGCC
/5AmMC6/CTACCATAGGGTAAAACCACT


(SEQ. ID. No. 48)
(SEQ. ID. No. 49)


 


/5AmMC6/CCTCTAAAAGGAAGCACTTTCT
/5AmMC6/TGGAGACACGTGCACTGTAGA


(SEQ. ID. No. 50)
(SEQ. ID. No. 51)


 


/5AmMC6/GAACATACAAAGGGTATCCTCT
/5AmMC6/TTCACATAGGAATAAAAAGCCATA


(SEQ. ID. No. 52)
(SEQ. ID. No. 53)


 


/5AmMC6/CGAATATAACACGGTCGATCT
/5AmMC6/ACAGCTGGTTGAAGGGGACCAA


(SEQ. ID. No. 54)
(SEQ. ID. No. 55)


 


/5AmMC6/CCTCCAGCCCCTCCAGGGCT
/5AmMC6/GGGGTATTTGACAAACTGACA


(SEQ. ID. No. 56)
(SEQ. ID. No. 57)


 


/5AmMC6/TCAATCACAGATAGCACCCCT
/5AmMC6/GCAAAAATGTGCTAGTGCCAAA


(SEQ. ID. No. 58)
(SEQ. ID. No. 59)


 


/5AmMC6/GTAGTGCAACTATGCAAAACT
/5AmMC6/TCATACAGCTAGATAACCAAAGA


(SEQ. ID. No. 60)
(SEQ. ID. No. 61)


 


/5AmMC6/TGGTGGCAGTGGTGGGAT
/5AmMC6/CTTCCAGTCGGGGATGTTTACA


(SEQ. ID. No. 62)
(SEQ. ID. No. 63)


 


/5AmMC6/ACACTCTAAAGGGATGCACGAT
/5AmMC6/ACACAAATTCGGTTCTACAGGG


(SEQ. ID. No. 64)
(SEQ. ID. No. 65)


 


/5AmMC6/AAAGTGCTTCTTACCTCCAGAT
/5AmMC6/ACCCTCCACCATGCAAGGGATG


(SEQ. ID. No. 66)
(SEQ. ID. No. 67)


 


/5AmMC6/ACCTCTAAAGGGGAGCGCTT
/5AmMC6/CCTATCTCCCCTCTGGACC


(SEQ. ID. No. 68)
(SEQ. ID. No. 69)


 


/5AmMC6/ACACTCTAAAGGGAGGCACTTT
/5AmMC6/GGCTGTCAATTCATAGGTCAG


(SEQ. ID. No. 70)
(SEQ. ID. No. 71)


 


/5AmMC6/TCCAGGAGCTCACAATCTAGTG
/5AmMC6/CGGCTGCAACACAAGACACGA


(SEQ. ID. No. 72)
(SEQ. ID. No. 73)


 


/5AmMC6/GTTACCGCAGGCTGCTCTGG
/5AmMC6/CAGTGAATTCTACCAGTGCCATA


(SEQ. ID. No. 74)
(SEQ. ID. No. 75)


 


/5AmMC6/AGAAAGCGCTTCCCTGTAGAG
/5AmMC6/TGTGAGTTCTACCATTGCCAAA


(SEQ. ID. No. 76)
(SEQ. ID. No. 77)


 


/5AmMC6/AGCCAAGTAATGGAGAACAGG
/5AmMC6/CGAAGGCAACACGGATAACCTA


(SEQ. ID. No. 78)
(SEQ. ID. No. 79)


 


/5AmMC6/AGAAAGCGCTTCCCTCTAGAG
/5AmMC6/TCACTTTTGTGACTATGCAA


(SEQ. ID. No. 80)
(SEQ. ID. No. 81)


 


/5AmMC6/ACAGAAAGGGCTTCCCTTTGC
/5AmMC6/CCAAGTTCTGTCATGCACTGA


(SEQ. ID. No. 82)
(SEQ. ID. No. 83)


 


/5AmMC6/TCGGTCCCTCGGGCCAGGG
/5AmMC6/GGAGTGAAGACACGGAGCCAGA


(SEQ. ID. No. 84)
(SEQ. ID. No. 85)


 


/5AmMC6/TCTAAGCCACCATGTGAAACCA
/5AmMC6/ACAAAGTTCTGTGATGCACTGA


(SEQ. ID. No. 86)
(SEQ. ID. No. 87)


 


/5AmMC6/GCAAGGCAGTGGCCTGTACA
/5AmMC6/GCTGAGAGTGTAGGATGTTTACA


(SEQ. ID. No. 88)
(SEQ. ID. No. 89)


 


/5AmMC6/TCCAGCAAAGGGAAGCGCTT
/5AmMC6/AACCGATTTCAGATGGTGCTAG


(SEQ. ID. No. 90)
(SEQ. ID. No. 91)


 


/5AmMC6/ACCCTCTATAGGGAAGCGCGT
/5AmMC6/GTCATCATTACCAGGCAGTATTA


(SEQ. ID. No. 92)
(SEQ. ID. No. 93)


 


/5AmMC6/CAGGTAACAACTCGCCGCTC
/5AmMC6/AACCAATGTGCAGACTACTGTA


(SEQ. ID. No. 94)
(SEQ. ID. No. 95)


 


/5AmMC6/ACTGCACTTTTATGAATAAGCTC
/5AmMC6/GCTGGGTGGAGAAGGTGGTGAA


(SEQ. ID. No. 96)
(SEQ. ID. No. 97)


 


/5AmMC6/AACGCTCCAAAAGAAGGCACT
/5AmMC6/GCCAATATTTCTGTGCTGCTA


(SEQ. ID. No. 98)
(SEQ. ID. No. 99)


 


/5AmMC6/ACCAGAACTGAGTCCACAGGG
/5AmMC6/CGCAAGGTCGGTTCTACGGGTG


(SEQ. ID. No. 100)
(SEQ. ID. No. 101)


 


/5AmMC6/TCCCGTCGCCAGCGGAGGC
/5AmMC6/ACACCGAGGAGCCCATCATGAT


(SEQ. ID. No. 102)
(SEQ. ID. No. 103)


 


/5AmMC6/ACTGAAACCAAGTATGGGTCGC
/5AmMC6/CCTGCATGACGGCCTGCAAGACA


(SEQ. ID. No. 104)
(SEQ. ID. No. 105)


 


/5AmMC6/CAGCCCTCCTGGTGGCTGG
/5AmMC6/ATAAGGATTTTTAGGGGCATTA


(SEQ. ID. No. 106)
(SEQ. ID. No. 107)


 


/5AmMC6/ATCTACACTGGCTACTGAGCC
/5AmMC6/TATTAGGAACACATCGCAAAAA


(SEQ. ID. No. 108)
(SEQ. ID. No. 109)


 


/5AmMC6/CGCGACTGCGTCACCGGCC
/5AmMC6/ACCAGCTAACAATACACTGCCA


(SEQ. ID. No. 110)
(SEQ. ID. No. 111)


 


/5AmMC6/CCTGCGCCATCTCCTCTAC
/5AmMC6/ATGGGACATCCTACATATGCAA


(SEQ. ID. No. 112)
(SEQ. ID. No. 113)


 


/5AmMC6/ACTGTGTTTCAGCTCAGTAGGCA
/5AmMC6/TTCAAAACATGAATTGCTGCTG


(SEQ. ID. No. 114)
(SEQ. ID. No. 115)


 


/5AmMC6/CGAACACAGCAGGGATAACCAC
/5AmMC6/GTACCCCTGGAGATTCTGATAA


(SEQ. ID. No. 116)
(SEQ. ID. No. 117)


 


/5AmMC6/ACTGCAGAACTGTTCCCGCTG
/5AmMC6/TCACGCGAGCCGAACGAACAAA


(SEQ. ID. No. 118)
(SEQ. ID. No. 119)


 


/5AmMC6/AGAAAATGCCCCTCAGTTTTGA
/5AmMC6/ACAAAAGTTGCCTTTGTGTGAT


(SEQ. ID. No. 120)
(SEQ. ID. No. 121)


 


/5AmMC6/AGACGGGAGGAGAGGAGTGA
/5AmMC6/ACACAGGACCTGGAGTCAGGAG


(SEQ. ID. No. 122)
(SEQ. ID. No. 123)


 


/5AmMC6/ATCGGGAGGGGACTGAGCCT
/5AmMC6/CCTACGTTCCATAGTCTACCA


(SEQ. ID. No. 124)
(SEQ. ID. No. 125)


 


/5AmMC6/CTCGGGGCAGCTCAGTACAG
/5AmMC6/GCGCATGTTCTATGGTCAACCA


(SEQ. ID. No. 126)
(SEQ. ID. No. 127)


 


/5AmMC6/CGAGCCGGTCGAGGTCCGGT
/5AmMC6/ACAGAGAGCTTGCCCTTGTATA


(SEQ. ID. No. 128)
(SEQ. ID. No. 129)


 


/5AmMC6/TCTCGTGACATGATGATCCCCG
/5AmMC6/TATGAACAATTTCTAGGAAT


(SEQ. ID. No. 130)
(SEQ. ID. No. 131)


 


/5AmMC6/AGAAAGCGCTTTCCTTTGTAGA
/5AmMC6/GGCGGACACGACATTCCCGAT


(SEQ. ID. No. 132)
(SEQ. ID. No. 133)


 


/5AmMC6/CACATGGCCAAAACAGAGAAGA
/5AmMC6/GTCTCAGTTTCCTCTGCAAACA


(SEQ. ID. No. 134)
(SEQ. ID. No. 135)


 


/5AmMC6/CCACCCAATGACCTACTCCAAG
/5AmMC6/GCTGTAAACATCCGACTGAAAG


(SEQ. ID. No. 136)
(SEQ. ID. No. 137)


 


/5AmMC6/TCATCTCGCCCGCAAAGACC
/5AmMC6/ATGCTTTTTGGGGTAAGGGCTT


(SEQ. ID. No. 138)
(SEQ. ID. No. 139)


 


/5AmMC6/AGAAAGTGCTTTCTTTTGGAGAA
/5AmMC6/ACGGCATTACCAGACAGTATTA


(SEQ. ID. No. 140)
(SEQ. ID. No. 141)


 


/5AmMC6/GAAAGTGCTTCTTTCCTCGAGAA
/5AmMC6/TCTCTGCAGGCCCTGTGCTTTGC


(SEQ. ID. No. 142)
(SEQ. ID. No. 143)


 


/5AmMC6/CACAGGTTAAAGGGTCTCAGGGA
/5AmMC6/TGTTGCAGCGCTTCATGTTT


(SEQ. ID. No. 144)
(SEQ. ID. No. 145)


 


/5AmMC6/ACAAATTCGGTTCTACAGGGTA
/5AmMC6/AGCCACAGTCACCTTCTGATCT


(SEQ. ID. No. 146)
(SEQ. ID. No. 147)


 


/5AmMC6/TGATAGCCCTGTACAATGCTGCT
/5AmMC6/CATGCATACATGCACACATACAT


(SEQ. ID. No. 148) 
(SEQ. ID. No. 149)


 


/5AmMC6/TCATAGCCCTGTACAATGCTGCT
/5AmMC6/CAACAAACATTTAATGAGGCC


(SEQ. ID. No. 150) 
(SEQ. ID. No. 151)


 


/5AmMC6/AACTATACAATCTACTACCTCA
/5AmMC6/TGATGGACAACAAATTAGGTA


(SEQ. ID. No. 152)
(SEQ. ID. No. 153)


 


/5AmMC6/ACTATGCAACCTACTACCTCT
/5AmMC6/TATCTCACAGAATAAACTTGGTA


(SEQ. ID. No. 154)
(SEQ. ID. No. 155)


 


/5AmMC6/TTCAGCTATCACAGTACTGTA
/5AmMC6/TCACATCAGTGCCATTCTAAATA


(SEQ. ID. No. 156)
(SEQ. ID. No. 157)


 


/5AmMC6/CTATACAACCTCCTACCTCA
/5AmMC6/GTCTTATGTGTGCGTGTATGTAT


(SEQ. ID. No. 158)
(SEQ. ID. No. 159)


 


/5AmMC6/CTCAATAGACTGTGAGCTCCTT
/5AmMC6/GTGTAGGTGTGTGTATGTATAT


(SEQ. ID. No. 160)
(SEQ. ID. No. 161)


 


/5AmMC6/AACCTATCCTGAATTACTTGAA
/5AmMC6/CAGACACACGCACATCAGTCATA


(SEQ. ID. No. 162)
(SEQ. ID. No. 163)


 


/5AmMC6/TCCATCATCAAAACAAATGGAGT
/5AmMC6/GGACACCAAGATCAATGAAAGAGGCA


(SEQ. ID. No. 164) 
(SEQ. ID. No. 165)


 


/5AmMC6/AGGCAAAGGATGACAAAGGGAA
/5AmMC6/TCACCAGTGCCAGTCCAAGAA


(SEQ. ID. No. 166)
(SEQ. ID. No. 167)


 


/5AmMC6/GAACAGGTAGTCTAAACACTGGG
/5AmMC6/TGTGAAAAGCACTATACTACGTA


(SEQ. ID. No. 168)
(SEQ. ID. No. 169)


 


/5AmMC6/ATCCAGTCAGTTCCTGATGCAGTA
/5AmMC6/AGACTAGATATGGAAGGGTGA


(SEQ. ID. No. 170)
(SEQ. ID. No. 171)


 


/5AmMC6/GCTGCAAACATCCGACTGAAAG
/5AmMC6/TCTGGGCACACGGAGGGAGA


(SEQ. ID. No. 172)
(SEQ. ID. No. 173)


 


/5AmMC6/TAACCGATTTCAAATGGTGCTA
/5AmMC6/ACGGTCAGGCTTTGGCTAGAT


(SEQ. ID. No. 174)
(SEQ. ID. No. 175)


 


/5AmMC6/AACAATACAACTTACTACCTCA
/5AmMC6/AGAGGCAGGCACTCAGGCAGA


(SEQ. ID. No. 176)
(SEQ. ID. No. 177)


 


/5AmMC6/ATACATACTTCTTTACATTCCA
/5AmMC6/TGGGCGACCCAGAGGGACA


(SEQ. ID. No. 178)
(SEQ. ID. No. 179)


 


/5AmMC6/GCTGAGTGTAGGATGTTTACA
/5AmMC6/AGAGGTTAAGACAGCAGGGCTG


(SEQ. ID. No. 180)
(SEQ. ID. No. 181)


 


/5AmMC6/ATGCCCTTTTAACATTGCACTG
/5AmMC6/GGTTCAAACCATGAGTCGAGCT


(SEQ. ID. No. 182)
(SEQ. ID. No. 183)


 


/5AmMC6/CTACGCGTATTCTTAAGCAATAA
/5AmMC6/GGAGTCGAGTGATGGTTCAAA


(SEQ. ID. No. 184)
(SEQ. ID. No. 185)


 


/5AmMC6/AGAACAATGCCTTACTGAGTA
/5AmMC6/GAATAATGACAGGCTCACCGTA


(SEQ. ID. No. 186)
(SEQ. ID. No. 187)


 


/5AmMC6/TCTTCCCATGCGCTATACCTCT
/5AmMC6/TACTATGCAACCTACTACTCT


(SEQ. ID. No. 188)
(SEQ. ID. No. 189)


 


/5AmMC6/CCACACACTTCCTTACATTCCA
/5AmMC6/CTGAGGGGCCTCAGACCGAGCT


(SEQ. ID. No. 190)
(SEQ. ID. No. 191)


 


/5AmMC6/GAGGGAGGAGAGCCAGGAGAAGC
/5AmMC6/TAACTGCACTAGATGCACCTTA


(SEQ. ID. No. 192)
(SEQ. ID. No. 193)


 


/5AmMC6/ACAAGCTTTTTGCTCGTCTTAT
/5AmMC6/CTACCTGCACTATGAGCACTTTG


(SEQ. ID. No. 194)
(SEQ. ID. No. 195)


 


/5AmMC6/GGCCGTGACTGGAGACTGTTA
/5AmMC6/GGGGACGAAATCCAAGCGCAGC


(SEQ. ID. No. 196)
(SEQ. ID. No. 197)


 


/5AmMC6/CACAGTTGCCAGCTGAGATTA
/5AmMC6/AATAGGTCAACCGTGTATGATT


(SEQ. ID. No. 198)
(SEQ. ID. No. 199)


 


/5AmMC6/ACATGGTTAGATCAAGCACAA
/5AmMC6/GCAGAAGCATTTCCACACAC


(SEQ. ID. No. 200)
(SEQ. ID. No. 201)


 


/5AmMC6/ACAACCAGCTAAGACACTGCCA
/5AmMC6/CCCCTATCACGATTAGCATTAA


(SEQ. ID. No. 202)
(SEQ. ID. No. 203)


 


/5AmMC6/AGGCGAAGGATGACAAAGGGAA
/5AmMC6/ACAAGTGCCTTCACTGCAGT


(SEQ. ID. No. 204)
(SEQ. ID. No. 205)


 


/5AmMC6/GTCTGTCAATTCATAGGTCAT
/5AmMC6/TCCAGCACTGTCCGGTAAGATG


(SEQ. ID. No. 206)
(SEQ. ID. No. 207)


 


/5AmMC6/ATCCAATCAGTTCCTGATGCAGTA
/5AmMC6/AAAGCAAGTACATCCACGTTTA


(SEQ. ID. No. 208)
(SEQ. ID. No. 209)


 


/5AmMC6/ACTACCTGCACTGTAAGCACTTTG
/5AmMC6/AAGCGGTTTACCATCCCACATA


(SEQ. ID. No. 210)
(SEQ. ID. No. 211)


 


/5AmMC6/TATCTGCACTAGATGCACCTTA
/5AmMC6/TAGTTGGCAAGTCTAGAACCA


(SEQ. ID. No. 212)
(SEQ. ID. No. 213)


 


/5AmMC6/AACCCACCGACAGCAATGAATGTT
/5AmMC6/CTACTAAAACATGGAAGCACTTA


(SEQ. ID. No. 214)
(SEQ. ID. No. 215)


 


/5AmMC6/GAACAGGTAGTCTGAACACTGGG
/5AmMC6/AGAAAGCACTTCCATGTTAAAGT


(SEQ. ID. No. 216)
(SEQ. ID. No. 217)


 


/5AmMC6/GAACAGATAGTCTAAACACTGGG
/5AmMC6/CCACTGAAACATGGAAGCACTTA


(SEQ. ID. No. 218)
(SEQ. ID. No. 219)


 


/5AmMC6/TCAGTTTTGCATAGATTTGCACA
/5AmMC6/CAGCAGGTACCCCCATGTTA


(SEQ. ID. No. 220)
(SEQ. ID. No. 221)


 


/5AmMC6/CTAGTGGTCCTAAACATTTCAC
/5AmMC6/ACACTCAAACATGGAAGCACTTA


(SEQ. ID. No. 222)
(SEQ. ID. No. 223)


 


/5AmMC6/AGGCATAGGATGACAAAGGGAA
/5AmMC6/ACTTACTGGACACCTACTAGG


(SEQ. ID. No. 224)
(SEQ. ID. No. 225)


 


/5AmMC6/CAGCCGCTGTCACACGCACAG
/5AmMC6/AAAGGCATCATATAGGAGCTGGA


(SEQ. ID. No. 226)
(SEQ. ID. No. 227)


 


/5AmMC6/CTGCCTGTCTGTGCCTGCTGT
/5AmMC6/GCCCTGGACTAGGAGTCAGCA


(SEQ. ID. No. 228)
(SEQ. ID. No. 229)


 


/5AmMC6/CACAAGTTCGGATCTACGGGTT
/5AmMC6/AGAGGCAGGCATGCGGGCAG


(SEQ. ID. No. 230)
(SEQ. ID. No. 231)


 


/5AmMC6/GCTACCTGCACTGTAAGCACTTTT
/5AmMC6/TCACCATTGCTAAAGTGCAATT


(SEQ. ID. No. 232)
(SEQ. ID. No. 233)


 


/5AmMC6/CACAAATTCGGATCTACAGGGTA
/5AmMC6/AAACGTGGAATTTCCTCTATGT


(SEQ. ID. No. 234)
(SEQ. ID. No. 235)


 


/5AmMC6/ACAAACACCATTGTCACACTCCA
/5AmMC6/AAAGATCAACCATGTATTATT


(SEQ. ID. No. 236)
(SEQ. ID. No. 237)


 


/5AmMC6/CGCGTACCAAAAGTAATAATG
/5AmMC6/AGCCACAATCACCTTCTGATCT


(SEQ. ID. No. 238)
(SEQ. ID. No. 239)


 


/5AmMC6/GCCCTTTCATCATTGCACTG
/5AmMC6/TCTCTGCAGGCCGTGTGCTTTGC


(SEQ. ID. No. 240)
(SEQ. ID. No. 241)


 


/5AmMC6/TCCCTCTGGTCAACCAGTCACA
/5AmMC6/ACGGTTTTACCAGACAGTATTA


(SEQ. ID. No. 242)
(SEQ. ID. No. 243)


 


/5AmMC6/GTAGTGCTTTCTACTTTATG
/5AmMC6/ACGTGGATTTTCCTCTATGAT


(SEQ. ID. No. 244)
(SEQ. ID. No. 245)


 


/5AmMC6/CCCCTATCACAATTAGCATTAA
/5AmMC6/GGCCTTCTGACTCCAAGTCCAG


(SEQ. ID. No. 246)
(SEQ. ID. No. 247)


 


/5AmMC6/TGTAAACCATGATGTGCTGCTA
/5AmMC6/AAGATGTGGACCATATTACATA


(SEQ. ID. No. 248)
(SEQ. ID. No. 249)


 


/5AmMC6/ACTCACCGACAGGTTGAATGTT
/5AmMC6/GGCCTTCTGACCCTAAGTCCAG


(SEQ. ID. No. 250)
(SEQ. ID. No. 251)


 


/SAmMC6/CACAAACCATTATGTGCTGCTA
/SAmMC6/AAAGAGGTTAACCAGGTGTGTT


(SEQ. ID. No. 252)
(SEQ. ID. No. 253)


 


/5AmMC6/TAACTGTACAAACTACTACCTCA
/5AmMC6/CGAACTCACCACGGACAACCTC


(SEQ. ID. No. 254)
(SEQ. ID. No. 255)


 


/5AmMC6/ACAGGCCGGGACAAGTGCAATAT
/5AmMC6/CTTCTTTGCAGATGAGACTGA


(SEQ. ID. No. 256)
(SEQ. ID. No. 257)


 


/5AmMC6/TAACCCATGGAATTCAGTTCTCA
/5AmMC6/TGCAAAGTTGCTCGGGTAACCT


(SEQ. ID. No. 258)
(SEQ. ID. No. 259)


 


/SAmMC6/AACCATACAACCTACTACCTCA
/SAmMC6/AACATGGATTTTCCTCTATGAT


(SEQ. ID. No. 260)
(SEQ. ID. No. 261)


 


/SAmMC6/AACAACAAAATCACTAGTCTTCCA
/SAmMC6/GAATTCATCACGGCCAGCCTCT


(SEQ. ID. No. 262)
(SEQ. ID. No. 263)


 


/5AmMC6/ACTTTCGGTTATCTAGCTTTAT
/5AmMC6/AGAGGAGAGCCGTGTATGAC


(SEQ. ID. No. 264)
(SEQ. ID. No. 265)


 


/5AmMC6/ACAGCACAAACTACTACCTCA
/5AmMC6/TTGAGAGTGCCATTATCTGGG


(SEQ. ID. No. 266)
(SEQ. ID. No. 267)


 


/5AmMC6/AACTATACAACCTACTACCTCA
/5AmMC6/GCTGCCGTATATGTGATGTCACT


(SEQ. ID. No. 268)
(SEQ. ID. No. 269)


 


/5AmMC6/AACCACACAACCTACTACCTCA
/5AmMC6/CAGCATGGAGTCCTCCAGGTTG


(SEQ. ID. No. 270)
(SEQ. ID. No. 271)


 


/5AmMC6/CCGACCATGGCTGTAGACTGTTA
/5AmMC6/TCCTCATGGAAGGGTTCCCCACT


(SEQ. ID. No. 272)
(SEQ. ID. No. 273)


 


/5AmMC6/ACACCAATGCCCTAGGGGATGCG
/5AmMC6/TGACTGCAGAGCAAAAGACAC


(SEQ. ID. No. 274)
(SEQ. ID. No. 275)


 


/5AmMC6/TGGCATTCACCGCGTGCCTTA
/5AmMC6/AGCCTATGGAATTCAGTTCTCA


(SEQ. ID. No. 276)
(SEQ. ID. No. 277)


 


/5AmMC6/TCACAAGTTAGGGTCTCAGGGA
/5AmMC6/AAAGAAGTATATGCATAGGAAA


(SEQ. ID. No. 278)
(SEQ. ID. No. 279)


 


/5AmMC6/CACAAGATCGGATCTACGGGT
/5AmMC6/TTTTCCCATGCCCTATACCTCT


(SEQ. ID. No. 280)
(SEQ. ID. No. 281)


 


/5AmMC6/CGCCAATATTTACGTGCTGCTA
/5AmMC6/AAGAATCTTGTCCCGCAGGTCCT


(SEQ. ID. No. 282)
(SEQ. ID. No. 283)


 


/5AmMC6/AACACTGATTTCAAATGGTGCTA
/5AmMC6/AATGAAAGCCTACCATGTACAA


(SEQ. ID. No. 284)
(SEQ. ID. No. 285)


 


/5AmMC6/CTTCAGTTATCACAGTACTGTA
/5AmMC6/AGACATGGAGGAGCCATCCAG


(SEQ. ID. No. 286)
(SEQ. ID. No. 287)


 


/5AmMC6/ACAGGAGTCTGAGCATTTGA
/5AmMC6/AAGAGGTTTCCCGTGTATGTTTCA


(SEQ. ID. No. 288)
(SEQ. ID. No. 289)


 


/5AmMC6/ATCTGCACTGTCAGCACTTTA
/5AmMC6/GGAGATTGGCCATGTAATACT


(SEQ. ID. No. 290)
(SEQ. ID. No. 291)


 


/5AmMC6/GCATTATTACTCACGGTACGA
/5AmMC6/ACAAACCACAGTGTGCTGCTG


(SEQ. ID. No. 292)
(SEQ. ID. No. 293)


 


/5AmMC6/AGCCAAGCTCAGACGGATCCGA
/5AmMC6/AACCCACCGACAACAATGAATGTT


(SEQ. ID. No. 294)
(SEQ. ID. No. 295)


 


/5AmMC6/ACTGATATCAGCTCAGTAGGCAC
/5AmMC6/GAAAGTGCCCTCAAGGCTGAGTG


(SEQ. ID. No. 296)
(SEQ. ID. No. 297)


 


/5AmMC6/TCCATCATTACCCGGCAGTATTA
/5AmMC6/GACCTCAGCTATGACAGCACTT


(SEQ. ID. No. 298)
(SEQ. ID. No. 299)


 


/5AmMC6/TAAACGGAACCACTAGTGACTTG
/5AmMC6/GAAAAACGCCCCCTGGCTTGAAA


(SEQ. ID. No. 300)
(SEQ. ID. No. 301)


 


/5AmMC6/TCAGACCGAGACAAGTGCAATG
/5AmMC6/CCCTCAAAAAGGAAGCACTTT


(SEQ. ID. No. 302)
(SEQ. ID. No. 303)


 


/5AmMC6/GGCGGAACTTAGCCACTGTGAA
/5AmMC6/GAAAGTGCTCCCTTTTGGAGAA


(SEQ. ID. No. 304)
(SEQ. ID. No. 305)


 


/5AmMC6/ACAGGATTGAGGGGGGGCCCT
/5AmMC6/ACACTCTAAAAGGAGGCACTTT


(SEQ. ID. No. 306)
(SEQ. ID. No. 307)


 


/5AmMC6/ATGTATGTGGGACGGTAAACCA
/5AmMC6/ATCCTCTAAAAAGATGCACTTT


(SEQ. ID. No. 308)
(SEQ. ID. No. 309)


 


/5AmMC6/GCTTTGACAATACTATTGCACTG
/5AmMC6/AGAAAGTACTTCCCTCTGGAG


(SEQ. ID. No. 310)
(SEQ. ID. No. 311)


 


/5AmMC6/TCACCAAAACATGGAAGCACTTA
/5AmMC6/ACAGTCCAAAGGGAAGCACTTT


(SEQ. ID. No. 312)
(SEQ. ID. No. 313)


 


/5AmMC6/GCTTCCAGTCGAGGATGTTTACA
/5AmMC6/AACAGAAAGTGCTTCCCTCAAGAG


(SEQ. ID. No. 314)
(SEQ. ID. No. 315)


 


/5AmMC6/TCCAGTCAAGGATGTTTACA
/5AmMC6/GCCTCTAAAAGGAAGCACTTT


(SEQ. ID. No. 316)
(SEQ. ID. No. 317)


 


/5AmMC6/CAGCTATGCCAGCATCTTGCCT
/5AmMC6/AAACCTCTAAAAGGATGCACTTT


(SEQ. ID. No. 318)
(SEQ. ID. No. 319)


 


/5AmMC6/GCAACTTAGTAATGTGCAATA
/5AmMC6/AGAAAGTGCATCCCTCTGGAG


(SEQ. ID. No. 320)
(SEQ. ID. No. 321)


 


/5AmMC6/CAATCAGCTAATGACACTGCCT
/5AmMC6/GCTCTAAAGGGAAGCGCCTTC


(SEQ. ID. No. 322)
(SEQ. ID. No. 323)


 


/5AmMC6/GCAATCAGCTAACTACACTGCCT
/5AmMC6/AGAGAAAGTGCTTCCCTCTAGAG


(SEQ. ID. No. 324)
(SEQ. ID. No. 325)


 


/5AmMC6/CTACCTGCACGAACAGCACTTTG
/5AmMC6/TCCTCTAAAGAGAAGCGCTTT


(SEQ. ID. No. 326)
(SEQ. ID. No. 327)


 


/5AmMC6/TGCTCAATAAATACCCGTTGAA
/5AmMC6/CAGAAAGTGCTTCCCTCCAGAGA


(SEQ. ID. No. 328)
(SEQ. ID. No. 329)


 


/5AmMC6/AGCAAGCCCAGACCGCAAAAAG
/5AmMC6/CACTCTAAAGAGAAGCGCTTTG


(SEQ. ID. No. 330)
(SEQ. ID. No. 331)


 


/5AmMC6/AGAAAGGCAGCAGGTCGTATAG
/5AmMC6/GAGAAAGTGCTTCCCTTTGTAG


(SEQ. ID. No. 332)
(SEQ. ID. No. 333)


 


/5AmMC6/TACCTGCACTGTTAGCACTTTG
/5AmMC6/ACTCCAAAGGGAAGCGCCTTC


(SEQ. ID. No. 334)
(SEQ. ID. No. 335)


 


/5AmMC6/CACATAGGAATGAAAAGCCATA
/5AmMC6/AGACAGTGCTTCCATCTAGAGG


(SEQ. ID. No. 336)
(SEQ. ID. No. 337)


 


/5AmMC6/CCTCAAGGAGCCTCAGTCTAGT
/5AmMC6/CAGAAAGGGCTTCCCTTTGTAGA


(SEQ. ID. No. 338)
(SEQ. ID. No. 339)


 


/5AmMC6/ACAAGTGCCCTCACTGCAGT
/5AmMC6/AACCCACCAAAGAGAAGCACTTT


(SEQ. ID. No. 340)
(SEQ. ID. No. 341)


 


/5AmMC6/TAAACGGAACCACTAGTGACTTA
/5AmMC6/ACACTCTAAAGGGAAGCACTTTGT


(SEQ. ID. No. 342)
(SEQ. ID. No. 343)


 


/5AmMC6/AAAAAGTGCCCCCATAGTTTGAG
/5AmMC6/AACCCTCTGAAAGGAAGCACTT


(SEQ. ID. No. 344)
(SEQ. ID. No. 345)


 


/5AmMC6/GGCACACAAAGTGGAAGCACTTT
/5AmMC6/GCTCCAAAGGGAAGCGCTTTG


(SEQ. ID. No. 346)
(SEQ. ID. No. 347)


 


/5AmMC6/AGAGAGGGCCTCCACTTTGATG
/5AmMC6/AAAGGGCTTCCCTTTGCAGA


(SEQ. ID. No. 348)
(SEQ. ID. No. 349)


 


/5AmMC6/ACACTCAAAACCTGGCGGCACTT
/5AmMC6/ACACTCTAAAAGGATGCACGAT


(SEQ. ID. No. 350)
(SEQ. ID. No. 351)


 


/5AmMC6/CAAAAGAGCCCCCAGTTTGAGT
/5AmMC6/TTAAACATCACTGCAAGTCTTAA


(SEQ. ID. No. 352)
(SEQ. ID. No. 353)


 


/5AmMC6/ACACTACAAACTCTGCGGCACT
/5AmMC6/CAGAATCCTTGCCCAGGTGCAT


(SEQ. ID. No. 354)
(SEQ. ID. No. 355)


 


/5AmMC6/ACACACAAAAGGGAAGCACTTT
/5AmMC6/TCTCACCCAGGGACAAAGGATT


(SEQ. ID. No. 356)
(SEQ. ID. No. 357)


 


/5AmMC6/AGACTCAAAAGTAGTAGCACTTT
/5AmMC6/TAGCACCCAGATAGCAAGGAT


(SEQ. ID. No. 358)
(SEQ. ID. No. 359)


 


/5AmMC6/CATGCACATGCACACATACAT
/5AmMC6/CTGCAGAACTGTTCCCGCTGCTA


(SEQ. ID. No. 360)
(SEQ. ID. No. 361)


 


/5AmMC6/GGAAGAACAGCCCTCCTCTGCC
/5AmMC6/ATAGAGTGCAGACCAGGGTCT


(SEQ. ID. No. 362)
(SEQ. ID. No. 363)


 


/5AmMC6/GAAGAGAGCTTGCCCTTGCATA
/5AmMC6/ATAAATGACACCTCCCTGTGAA


(SEQ. ID. No. 364)
(SEQ. ID. No. 365)


 


/5AmMC6/AGAGGTCGACCGTGTAATGTGC
/5AmMC6/TCTACTCAGAAGGGTGCCTTA


(SEQ. ID. No. 366)
(SEQ. ID. No. 367)


 


/5AmMC6/CCAGCAGCACCTGGGGCAGT
/5AmMC6/TTCACTCCAAAAGGTGCAAAA


(SEQ. ID. No. 368)
(SEQ. ID. No. 369)


 


/5AmMC6/ACACTTACTGAGCACCTACTAGG
/5AmMC6/TCTACTCCAAAAGGCTACAATCA


(SEQ. ID. No. 370)
(SEQ. ID. No. 371)


 


/5AmMC6/ACTGGAGGAAGGGCCCAGAGG
/5AmMC6/TCTACCCACAGACGTACCAATCA


(SEQ. ID. No. 372)
(SEQ. ID. No. 373)


 


/5AmMC6/ACGGAAGGGCAGAGAGGGCCAG
/5AmMC6/TGTGATTGCCACTCTCCTGAGTA


(SEQ. ID. No. 374)
(SEQ. ID. No. 375)


 


/5AmMC6/AAAAAGGTTAGCTGGGTGTGTT
/5AmMC6/CTACTCACAGAAGTGTCAAT


(SEQ. ID. No. 376)
(SEQ. ID. No. 377)


 


/5AmMC6/TTCTAGGATAGGCCCAGGGGC
/5AmMC6/TTCAATTTCTGCCGCAAAAG


(SEQ. ID. No. 378)
(SEQ. ID. No. 379)


 


/5AmMC6/AAAGGCATCATATAGGAGCTGAA
/5AmMC6/GCTATCTGCTGCAACAGAATTT


(SEQ. ID. No. 380)
(SEQ. ID. No. 381)


 


/5AmMC6/GGCTATAAAGTAACTGAGACGGA
/5AmMC6/GTGTGCTTACACACTTCCCGTTA


(SEQ. ID. No. 382)
(SEQ. ID. No. 383)


 


/5AmMC6/ACTGACCGACCGACCGATCGA
/5AmMC6/AGCACGTCACTTCCACTAAGA


(SEQ. ID. No. 384)
(SEQ. ID. No. 385)


 


/5AmMC6/ACAGTCAGGCTTTGGCTAGATCA
/5AmMC6/GCAAGGGCGAATGCAGAAAA


(SEQ. ID. No. 386)
(SEQ. ID. No. 387)


 


/5AmMC6/GCACTGGACTAGGGGTCAGCA
/5AmMC6/AACTCCGGGGCTGATCAGGT


(SEQ. ID. No. 388)
(SEQ. ID. No. 389)


 


/5AmMC6/AGAGGCAGGCACTCGGGCAGA
/5AmMC6/CTTGTACCAGTTATCTGCAA


(SEQ. ID. No. 390)
(SEQ. ID. No. 391)


 


/5AmMC6/CAATCAGCTAATTACACTGCCTA
/5AmMC6/TTGTACGTTTACATGGAGGTC


(SEQ. ID. No. 392)
(SEQ. ID. No. 393)


 


/5AmMC6/GTGAAAGTGTATGGGCTTTGTGAA
/5AmMC6/CTGACTGACTGACTGACTGACTG


(SEQ. ID. No. 394) 
(SEQ. ID. No. 395)


 


/5AmMC6/CAGGCTCAAAGGGCTCCTCAGG
/5AmMC6/CCATAAAGTAGGAAACACTA


(SEQ. ID. No. 396)
(SEQ. ID. No. 397)


 


/5AmMC6/AACAAAATCACAAGTCTTCCA
/5AmMC6/TCACCGACAGCGTTGAATGT


(SEQ. ID. No. 398)
(SEQ. ID. No. 399)


 


/5AmMC6/TGTAAGTGCTCGTAATGCAGT
/5AmMC6/CGGGACTTTGAGGGCCAGT


(SEQ. ID. No. 400)
(SEQ. ID. No. 401)


 


/5AmMC6/ACCCTCATGCCCCTCAAGG
/5AmMC6/GAATCCACCACGAACAACTT


(SEQ. ID. No. 402)
(SEQ. ID. No. 403)


 


/5AmMC6/AAAAGTAACTAGCACACCAC
/5AmMC6/AGAGACCGGTTCACTGTGA


(SEQ. ID. No. 404)
(SEQ. ID. No. 405)


 


/5AmMC6/ACATTTTTCGTTATTGCTCTT
/5AmMC6/AGAGACCGGTTCACTGTGA


(SEQ. ID. No. 406)
(SEQ. ID. No. 407)


 


/5AmMC6/TATGGCAGACTGTGATTTGTTG
/5AmMC6/CCTGATTCACAACACCAGCT


(SEQ. ID. No. 408)
(SEQ. ID. No. 409)


 


/5AmMC6/CATCGTTACCAGACAGTGTTA
/5AmMC6/GGATTCCTGGGAAAACTGGA


(SEQ. ID. No. 410)
(SEQ. ID. No. 411)


 


/5AmMC6/TCCACATGGAGTTGCTGTTACA
/5AmMC6/ACTGGTACAAGGGTTGGGAG


(SEQ. ID. No. 412)
(SEQ. ID. No. 413)


 


/5AmMC6/AACAGCTGCTTTTGGGATTCTG
/5AmMC6/CTGGGACTTTGTAGGCCAGT


(SEQ. ID. No. 414)
(SEQ. ID. No. 415)


 


/5AmMC6/ACCTAATATATCAAACATATCA
/5AmMC6/AGACTCCGGTGGAATGAAGG


(SEQ. ID. No. 416)
(SEQ. ID. No. 417)


 


/5AmMC6/AAGCCCAAAAGGAGAATTCTTTG
/5AmMC6/CAACATCAGTCTGATAAGCTA


(SEQ. ID. No. 418)
(SEQ. ID. No. 419)


 


/5AmMC6/AGGAACTGCCTTTCTCTCCAA
/5AmMC6/GTACAATCAACGGTCGATGG


(SEQ. ID. No. 420)
(SEQ. ID. No. 421)


 


/5AmMC6/ACCCTTATCAGTTCTCCGTCCA
/5AmMC6/AGAATTGCGTTTGGACAATC


(SEQ. ID. No. 422)
(SEQ. ID. No. 423)


 


/5AmMC6/TAGCTGGTTGAAGGGGACCAA
/5AmMC6/ACCCAGCAGACAATGTAGC


(SEQ. ID. No. 424)
(SEQ. ID. No. 425)


 


/5AmMC6/CCTCAAGGAGCTTCAGTCTAGT
/5AmMC6/ACCCAGTAGCCAGATGTAGC


(SEQ. ID. No. 426)
(SEQ. ID. No. 427)


 


/5AmMC6/CCAACAACAGGAAACTACCTA
/5AmMC6/GCCCTCTCAACCCAGCTTT


(SEQ. ID. No. 428)
(SEQ. ID. No. 429)


 


/5AmMC6/CCAGGTTCCACCCCAGCAGG
/5AmMC6/GCAATGCAACTACAATGCAC


(SEQ. ID. No. 430)
(SEQ. ID. No. 431)


 


/5AmMC6/ACACTCAAAAGATGGCGGCA
/5AmMC6/AACAAAATCACTGATGCTGG


(SEQ. ID. No. 432)
(SEQ. ID. No. 433)


 


/5AmMC6/ACGCTCAAATGTCGCAGCAC
/5AmMC6/GAGCTCCTGGAGGACAGGG


(SEQ. ID. No. 434)
(SEQ. ID. No. 435)


 


/5AmMC6/ACACCCCAAAATCGAAGCAC
/5AmMC6/GGGTGCGATTTCTGTGTGAG


(SEQ. ID. No. 436)
(SEQ. ID. No. 437)


 


/5AmMC6/GGAAAGCGCCCCCATTTTGA
/5AmMC6/ACTCAGTAATGGTAACGGTT


(SEQ. ID. No. 438)
(SEQ. ID. No. 439)


 


/5AmMC6/CACTTATCAGGTTGTATTATAA
/5AmMC6/GAGGAAACCAGCAAGTGTTG


(SEQ. ID. No. 440)
(SEQ. ID. No. 441)


 


/5AmMC6/GTCTGTCAAATCATAGGTCAT
/5AmMC6/GCAATGCAACAGCAATGCAC


(SEQ. ID. No. 442)
(SEQ. ID. No. 443)


 


/5AmMC6/GGGGTTCACCGAGCAACATTC
/5AmMC6/GTCCGTGGTTCTACCCTGTGG


(SEQ. ID. No. 444)
(SEQ. ID. No. 445)


 


/5AmMC6/CAGGCCATCTGTGTTATATT
/5AmMC6/ATACTAGACTGTGAGCTCCTCGA


(SEQ. ID. No. 446)
(SEQ. ID. No. 447)


 


/5AmMC6/AGTGGATGTTCCTCTATGAT
/5AmMC6/CCAGAAGGAGCACTTAGGGCAG


(SEQ. ID. No. 448)
(SEQ. ID. No. 449)


 


/5AmMC6/CGTGGATTTTCCTCTACGAT
/5AmMC6/GCTGGATGCAAACCTGCAAAAC


(SEQ. ID. No. 450)
(SEQ. ID. No. 451)


 


/5AmMC6/GAGGGTTAGTGGACCGTGTT
/5AmMC6/AATCCCATCCCCAGGAACCC


(SEQ. ID. No. 452)
(SEQ. ID. No. 453)


 


/5AmMC6/GATGTGGACCATACTACATA
/5AmMC6/CGGCTCTGTCGTCGAGGCGC


(SEQ. ID. No. 454)
(SEQ. ID. No. 455)


 


/5AmMC6/GGCTAGTGGACCAGGTGAAG
/5AmMC6/AAAGTCTCGCTCTCTGCCCCT


(SEQ. ID. No. 456)
(SEQ. ID. No. 457)


 


/5AmMC6/CAGAACTTAGCCACTGTGAA
/5AmMC6/TCAACGGGAGTGATCGTGTCAT


(SEQ. ID. No. 458)
(SEQ. ID. No. 459)


 


/5AmMC6/AGCCTATCCTGGATTACTTGAA
/5AmMC6/AGCATTGCAACCGATCCCAAC


(SEQ. ID. No. 460)
(SEQ. ID. No. 461)


 


/5AmMC6/CTGTTCCTGCTGAACTGAGCCA
/5AmMC6/GCAGCAAACATCTGACTGAAAG


(SEQ. ID. No. 462)
(SEQ. ID. No. 463)
















TABLE 5







miRNA detection probes


ProbeID: Unique identifier for each probe sequence.


Annotation: miRNAs recognized by the probe, based on miRBASE release


7.0. “h” stands for human, “m” for mouse and “r” for rat.


Sequence: sequence of probe. /5AmMC6/ indicates 5′ amino modification.









Probe
Annotation
Sequence













EAM190
h-miR-10b_rfam7.0
/5AmMC6/ACAAATTCGGTTCTACAGGGTA
(SEQ. ID. No. 464)


 


EAM187
hmr-miR-107_rfam7.0
/5AmMC6/TGATAGCCCTGTACAATGCTGCT
(SEQ. ID. No. 465)


 


EAM185
hmr-miR-103_rfam7.0
/5AmMC6/TCATAGCCCTGTACAATGCTGCT
(SEQ. ID. No. 466)


 


EAM181
hmr-let-7f_rfam7.0
/5AmMC6/AACTATACAATCTACTACCTCA
(SEQ. ID. No. 467)


 


EAM179
hmr-let-7d_rfam7.0
/5AmMC6/ACTATGCAACCTACTACCTCT
(SEQ. ID. No. 468)


 


EAM177
mr-miR-101b_rfam7.0
/5AmMC6/TTCAGCTATCACAGTACTGTA
(SEQ. ID. No. 469)


 


EAM175
hmr-miR-320_rfam7.0
/5AmMC6/TCGCCCTCTCAACCCAGCTTTT
(SEQ. ID. No. 470)


 


EAM168
hmr-let-7e_rfam7.0
/5AmMC6/CTATACAACCTCCTACCTCA
(SEQ. ID. No. 471)


 


EAM161
hmr-miR-28_rfam7.0
/5AmMC6/CTCAATAGACTGTGAGCTCCTT
(SEQ. ID. No. 472)


 


EAM160
hmr-miR-26b_rfam7.0
/5AmMC6/AACCTATCCTGAATTACTTGAA
(SEQ. ID. No. 473)


 


EAM155
hmr-miR-136_rfam7.0
/5AmMC6/TCCATCATCAAAACAAATGGAGT
(SEQ. ID. No. 474)


 


EAM283
mr-miR-211_rfam7.0
/5AmMC6/AGGCAAAGGATGACAAAGGGAA
(SEQ. ID. No. 475)


 


EAM282
m-miR-199b_rfam7.0
/5AmMC6/GAACAGGTAGTCTAAACACTGGG
(SEQ. ID. No. 476)


 


EAM281
mr-miR-217_rfam7.0
/5AmMC6/ATCCAGTCAGTTCCTGATGCAGTA
(SEQ. ID. No. 477)


 


EAM280
hmr-miR-30a-3p rfam7.0
/5AmMC6/GCTGCAAACATCCGACTGAAAG
(SEQ. ID. No. 478)


 


EAM279
hmr-miR-29c_rfam7.0
/5AmMC6/TAACCGATTTCAAATGGTGCTA
(SEQ. ID. No. 479)


 


EAM278
hmr-miR-98_rfam7.0
/5AmMC6/AACAATACAACTTACTACCTCA
(SEQ. ID. No. 480)


 


EAM238
hm-miR-1_rfam7.0
/5AmMC6/ATACATACTTCTTTACATTCCA
(SEQ. ID. No. 481)


 


EAM270
hmr-miR-30b_rfam7.0
/5AmMC6/GCTGAGTGTAGGATGTTTACA
(SEQ. ID. No. 482)


 


EAM159
hmr-miR-130a rfam7.0
/5AmMC6/ATGCCCTTTTAACATTGCACTG
(SEQ. ID. No. 483)


 


EAM163
hmr-miR-142-3p_rfam7.0
/5AmMC6/TCCATAAAGTAGGAAACACTACA
(SEQ. ID. No. 484)


 


EAM171
hmr-miR-137_rfam7.0
/5AmMC6/CTACGCGTATTCTTAAGCAATAA
(SEQ. ID. No. 485)


 


EAM306
m-miR-201_rfam7.0
/5AmMC6/AGAACAATGCCTTACTGAGTA
(SEQ. ID. No. 486)


 


EAM307
m-miR-202_rfam7.0
/5AmMC6/TCTTCCCATGCGCTATACCTCT
(SEQ. ID. No. 487)


 


EAM308
hmr-miR-206_rfam7.0
/5AmMC6/CCACACACTTCCTTACATTCCA
(SEQ. ID. No. 488)


 


EAM309
m-miR-207_rfam7.0
/5AmMC6/GAGGGAGGAGAGCCAGGAGAAGC
(SEQ. ID. No. 489)


 


EAM310
hmr-miR-208_rfam7.0
/5AmMC6/ACAAGCTTTTTGCTCGTCTTAT
(SEQ. ID. No. 490)


 


EAM247
hmr-miR-212_rfam7.0
/5AmMC6/GGCCGTGACTGGAGACTGTTA
(SEQ. ID. No. 491)


 


EAM251
hmr-miR-216_rfam7.0
/5AmMC6/CACAGTTGCCAGCTGAGATTA
(SEQ. ID. No. 492)


 


EAM253
hmr-miR-218_rfam7.0
/5AmMC6/ACATGGTTAGATCAAGCACAA
(SEQ. ID. No. 493)


 


EAM275
hmr-miR-34a_rfam7.0
/5AmMC6/ACAACCAGCTAAGACACTGCCA
(SEQ. ID. No. 494)


 


EAM246
h-miR-211_rfam7.0
/5AmMC6/AGGCGAAGGATGACAAAGGGAA
(SEQ. ID. No. 495)


 


EAM250
h-miR-215_rfam7.0
/5AmMC6/GTCTGTCAATTCATAGGTCAT
(SEQ. ID. No. 496)


 


EAM252
h-miR-217_rfam7.0
/5AmMC6/ATCCAATCAGTTCCTGATGCAGTA
(SEQ. ID. No. 497)


 


EAM224
hmr-miR-17-5p_rfam7.0
/5AmMC6/ACTACCTGCACTGTAAGCACTTTG
(SEQ. ID. No. 498)


 


EAM225
hmr-miR-18a_rfam7.0
/5AmMC6/TATCTGCACTAGATGCACCTTA
(SEQ. ID. No. 499)


 


EAM226
hmr-miR-181a_rfam7.0
/5AmMC6/ACTCACCGACAGCGTTGAATGTT
(SEQ. ID. No. 500)


 


EAM227
hmr-miR-181b_rfam7.0
/5AmMC6/AACCCACCGACAGCAATGAATGTT
(SEQ. ID. No. 501)


 


EAM234
hmr-miR-199a_rfam7.0
/5AmMC6/GAACAGGTAGTCTGAACACTGGG
(SEQ. ID. No. 502)


 


EAM235
h-miR-199b_rfam7.0
/5AmMC6/GAACAGATAGTCTAAACACTGGG
(SEQ. ID. No. 503)


 


EAM236
hmr-miR-19a_rfam7.0
/5AmMC6/TCAGTTTTGCATAGATTTGCACA
(SEQ. ID. No. 504)


 


EAM241
hmr-miR-203 rfam7.0
/5AmMC6/CTAGTGGTCCTAAACATTTCAC
(SEQ. ID. No. 505)


 


EAM242
hmr-miR-204_rfam7.0
/5AmMC6/AGGCATAGGATGACAAAGGGAA
(SEQ. ID. No. 506)


 


EAM243
hmr-miR-205_rfam7.0
/5AmMC6/CAGACTCCGGTGGAATGAAGGA
(SEQ. ID. No. 507)


 


EAM245
hmr-miR-210_rfam7.0
/5AmMC6/CAGCCGCTGTCACACGCACAG
(SEQ. ID. No. 508)


 


EAM249
hmr-miR-214_rfam7.0
/5AmMC6/CTGCCTGTCTGTGCCTGCTGT
(SEQ. ID. No. 509)


 


EAM184
hmr-miR-100 rfam7.0
/5AmMC6/CACAAGTTCGGATCTACGGGTT
(SEQ. ID. No. 510)


 


EAM186
h-miR-106a_rfam7.0
/5AmMC6/GCTACCTGCACTGTAAGCACTTTT
(SEQ. ID. No. 511)


 


EAM189
hmr-miR-10a_rfam7.0
/5AmMC6/CACAAATTCGGATCTACAGGGTA
(SEQ. ID. No. 512)


 


EAM191
hmr-miR-122a_rfam7.0
/5AmMC6/ACAAACACCATTGTCACACTCCA
(SEQ. ID. No. 513)


 


EAM192
hmr-miR-126*_rfam7.0
/5AmMC6/CGCGTACCAAAAGTAATAATG
(SEQ. ID. No. 514)


 


EAM198
hmr-miR-130b_rfam7.0
/5AmMC6/GCCCTTTCATCATTGCACTG
(SEQ. ID. No. 515)


 


EAM202
hmr-miR-134_rfam7.0
/5AmMC6/TCCCTCTGGTCAACCAGTCACA
(SEQ. ID. No. 516)


 


EAM209
hmr-miR-142-5p_rfam7.0
/5AmMC6/GTAGTGCTTTCTACTTTATG
(SEQ. ID. No. 517)


 


EAM221
m-miR-155_rfam7.0
/5AmMC6/CCCCTATCACAATTAGCATTAA
(SEQ. ID. No. 518)


 


EAM223
hmr-miR-15b_rfam7.0
/5AmMC6/TGTAAACCATGATGTGCTGCTA
(SEQ. ID. No. 519)


 


EAM228
hmr-miR-181c_rfam7.0
/5AmMC6/ACTCACCGACAGGTTGAATGTT
(SEQ. ID. No. 520)


 


EAM222
hm-miR-15a_rfam7.0
/5AmMC6/CACAAACCATTATGTGCTGCTA
(SEQ. ID. No. 521)


 


EAM111
hm-let-7g_rfam7.0
/5AmMC6/TAACTGTACAAACTACTACCTCA
(SEQ. ID. No. 522)


 


EAM131
hmr-miR-92_rfam7.0
/5AmMC6/ACAGGCCGGGACAAGTGCAATAT
(SEQ. ID. No. 523)


 


EAM139
hmr-miR-146a_rfam7.0
/5AmMC6/TAACCCATGGAATTCAGTTCTCA
(SEQ. ID. No. 524)


 


EAM145
hmr-let-7c_rfam7.0
/5AmMC6/AACCATACAACCTACTACCTCA
(SEQ. ID. No. 525)


 


EAM109
hmr-miR-7_rfam7.0
/5AmMC6/AACAACAAAATCACTAGTCTTCCA
(SEQ. ID. No. 526)


 


EAM152
hm-miR-9*_rfam7.0
/5AmMC6/ACTTTCGGTTATCTAGCTTTAT
(SEQ. ID. No. 527)


 


JLA215
hmr-let-7i_rfam7.0
/5AmMC6/ACAGCACAAACTACTACCTCA
(SEQ. ID. No. 528)


 


EAM153
hmr-let-7a rfam7.0
/5AmMC6/AACTATACAACCTACTACCTCA
(SEQ. ID. No. 529)


 


EAM147
hmr-let-7b_rfam7.0
/5AmMC6/AACCACACAACCTACTACCTCA
(SEQ. ID. No. 530)


 


EAM137
hmr-miR-132_rfam7.0
/5AmMC6/CCGACCATGGCTGTAGACTGTTA
(SEQ. ID. No. 531)


 


EAM133
hmr-miR-324-5p_rfam7.0
/5AmMC6/ACACCAATGCCCTAGGGGATGCG
(SEQ. ID. No. 532)


 


EAM103
hmr-miR-124a_rfam7.0
/5AmMC6/TGGCATTCACCGCGTGCCTTA
(SEQ. ID. No. 533)


 


EAM105
hmr-miR-125b rfam7.0
/5AmMC6/TCACAAGTTAGGGTCTCAGGGA
(SEQ. ID. No. 534)


 


EAM121
hmr-miR-99a_rfam7.0
/5AmMC6/CACAAGATCGGATCTACGGGT
(SEQ. ID. No. 535)


 


EAM115
hmr-miR-16_rfam7.0
/5AmMC6/CGCCAATATTTACGTGCTGCTA
(SEQ. ID. No. 536)


 


EAM119
hmr-miR-29b_rfam7.0
/5AmMC6/AACACTGATTTCAAATGGTGCTA
(SEQ. ID. No. 537)


 


EAM311
hmr-miR-101_rfam7.0
/5AmMC6/CTTCAGTTATCACAGTACTGTA
(SEQ. ID. No. 538)


 


EAM312
h-miR-105_rfam7.0
/5AmMC6/ACAGGAGTCTGAGCATTTGA
(SEQ. ID. No. 539)


 


EAM313
hmr-miR-106b_rfam7.0
/5AmMC6/ATCTGCACTGTCAGCACTTTA
(SEQ. ID. No. 540)


 


EAM314
hmr-miR-126_rfam7.0
/5AmMC6/GCATTATTACTCACGGTACGA
(SEQ. ID. No. 541)


 


EAM315
hmr-miR-127_rfam7.0
/5AmMC6/AGCCAAGCTCAGACGGATCCGA
(SEQ. ID. No. 542)


 


EAM320
hm-miR-189_rfam7.0
/5AmMC6/ACTGATATCAGCTCAGTAGGCAC
(SEQ. ID. No. 543)


 


JLA216
hmr-miR-200c_rfam7.0
/5AmMC6/TCCATCATTACCCGGCAGTATTA
(SEQ. ID. No. 544)


 


EAM323
h-miR-224_rfam7.0
/5AmMC6/TAAACGGAACCACTAGTGACTTG
(SEQ. ID. No. 545)


 


EAM324
hmr-miR-25_rfam7.0
/5AmMC6/TCAGACCGAGACAAGTGCAATG
(SEQ. ID. No. 546)


 


EAM386
r-miR-336_rfam7.0
/5AmMC6/AGACTAGATATGGAAGGGTGA
(SEQ. ID. No. 547)


 


JLA218
r-miR-343_rfam7.0
/5AmMC6/TCTGGGCACACGGAGGGAGA
(SEQ. ID. No. 548)


 


EAM388
r-miR-344_rfam7.0
/5AmMC6/ACGGTCAGGCTTTGGCTAGAT
(SEQ. ID. No. 549)


 


EAM338
h-miR-95_rfam7.0
/5AmMC6/TGCTCAATAAATACCCGTTGAA
(SEQ. ID. No. 550)


 


JLA214
hmr-miR-129_rfam7.0
/5AmMC6/AGCAAGCCCAGACCGCAAAAAG
(SEQ. ID. No. 551)


 


EAM340
mr-let-7d*_rfam7.0
/5AmMC6/AGAAAGGCAGCAGGTCGTATAG
(SEQ. ID. No. 552)


 


EAM341
m-miR-106a_rfam7.0
/5AmMC6/TACCTGCACTGTTAGCACTTTG
(SEQ. ID. No. 553)


 


EAM342
hmr-miR-135b_rfam7.0
/5AmMC6/CACATAGGAATGAAAAGCCATA
(SEQ. ID. No. 554)


 


EAM343
mr-miR-151 rfam7.0
/5AmMC6/CCTCAAGGAGCCTCAGTCTAGT
(SEQ. ID. No. 555)


 


EAM344
m-miR-17-3p_rfam7.0
/5AmMC6/ACAAGTGCCCTCACTGCAGT
(SEQ. ID. No. 556)


 


EAM345
m-miR-224_rfam7.0
/5AmMC6/TAAACGGAACCACTAGTGACTTA
(SEQ. ID. No. 557)


 


EAM346
mr-miR-290_rfam7.0
/5AmMC6/AAAAAGTGCCCCCATAGTTTGAG
(SEQ. ID. No. 558)


 


EAM347
mr-miR-291-3p_rfam7.0
/5AmMC6/GGCACACAAAGTGGAAGCACTTT
(SEQ. ID. No. 559)


 


EAM348
mr-miR-291-5p rfam7.0
/5AmMC6/AGAGAGGGCCTCCACTTTGATG
(SEQ. ID. No. 560)


 


EAM349
mr-miR-292-3p_rfam7.0
/5AmMC6/ACACTCAAAACCTGGCGGCACTT
(SEQ. ID. No. 561)


 


EAM350
mr-miR-292-5p_rfam7.0
/5AmMC6/CAAAAGAGCCCCCAGTTTGAGT
(SEQ. ID. No. 562)


 


EAM351
m-miR-293_rfam7.0
/5AmMC6/ACACTACAAACTCTGCGGCACT
(SEQ. ID. No. 563)


 


EAM352
m-miR-294_rfam7.0
/5AmMC6/ACACACAAAAGGGAAGCACTTT
(SEQ. ID. No. 564)


 


EAM353
m-miR-295_rfam7.0
/5AmMC6/AGACTCAAAAGTAGTAGCACTTT
(SEQ. ID. No. 565)


 


EAM354
m-miR-297_rfam7.0
/5AmMC6/CATGCACATGCACACATACAT
(SEQ. ID. No. 566)


 


EAM355
mr-miR-298_rfam7.0
/5AmMC6/GGAAGAACAGCCCTCCTCTGCC
(SEQ. ID. No. 567)


 


EAM356
mr-miR-300_rfam7.0
/5AmMC6/GAAGAGAGCTTGCCCTTGCATA
(SEQ. ID. No. 568)


 


EAM358
hmr-miR-323_rfam7.0
/5AmMC6/AGAGGTCGACCGTGTAATGTGC
(SEQ. ID. No. 569)


 


EAM359
hmr-miR-324-3p_rfam7.0
/5AmMC6/CCAGCAGCACCTGGGGCAGT
(SEQ. ID. No. 570)


 


EAM360
mr-miR-325_rfam7.0
/5AmMC6/ACACTTACTGAGCACCTACTAGG
(SEQ. ID. No. 571)


 


EAM361
hmr-miR-326_rfam7.0
/5AmMC6/ACTGGAGGAAGGGCCCAGAGG
(SEQ. ID. No. 572)


 


EAM362
hmr-miR-328_rfam7.0
/5AmMC6/ACGGAAGGGCAGAGAGGGCCAG
(SEQ. ID. No. 573)


 


EAM363
mr-miR-329_rfam7.0
/5AmMC6/AAAAAGGTTAGCTGGGTGTGTT
(SEQ. ID. No. 574)


 


EAM365
hmr-miR-331_rfam7.0
/5AmMC6/TTCTAGGATAGGCCCAGGGGC
(SEQ. ID. No. 575)


 


EAM366
mr-miR-337_rfam7.0
/5AmMC6/AAAGGCATCATATAGGAGCTGAA
(SEQ. ID. No. 576)


 


EAM367
hmr-miR-338_rfam7.0
/5AmMC6/TCAACAAAATCACTGATGCTGGA
(SEQ. ID. No. 577)


 


EAM368
hmr-miR-339_rfam7.0
/5AmMC6/TGAGCTCCTGGAGGACAGGGA
(SEQ. ID. No. 578)


 


EAM369
hmr-miR-340 rfam7.0
/5AmMC6/GGCTATAAAGTAACTGAGACGGA
(SEQ. ID. No. 579)


 


EAM370
mr-miR-341_rfam7.0
/5AmMC6/ACTGACCGACCGACCGATCGA
(SEQ. ID. No. 580)


 


EAM371
hmr-miR-342_rfam7.0
/5AmMC6/GACGGGTGCGATTTCTGTGTGAGA
(SEQ. ID. No. 581)


 


EAM372
m-miR-344_rfam7.0
/5AmMC6/ACAGTCAGGCTTTGGCTAGATCA
(SEQ. ID. No. 582)


 


EAM373
mr-miR-345_rfam7.0
/5AmMC6/GCACTGGACTAGGGGTCAGCA
(SEQ. ID. No. 583)


 


EAM374
m-miR-346 rfam7.0
/5AmMC6/AGAGGCAGGCACTCGGGCAGA
(SEQ. ID. No. 584)


 


EAM375
mr-miR-34b_rfam7.0
/5AmMC6/CAATCAGCTAATTACACTGCCTA
(SEQ. ID. No. 585)


 


JLA217
mr-miR-350_rfam7.0
/5AmMC6/GTGAAAGGTATGGGCTTTGTGAA
(SEQ. ID. No. 586)


 


EAM377
mr-miR-351_rfam7.0
/5AmMC6/CAGGCTCAAAGGGCTCCTCAGG
(SEQ. ID. No. 587)


 


EAM378
mr-miR-7b_rfam7.0
/5AmMC6/AACAAAATCACAAGTCTTCCA
(SEQ. ID. No. 588)


 


EAM382
r-miR-20*_rfam7.0
/5AmMC6/TGTAAGTGCTCGTAATGCAGT
(SEQ. ID. No. 589)


 


EAM383
r-miR-327_rfam7.0
/5AmMC6/ACCCTCATGCCCCTCAAGG
(SEQ. ID. No. 590)


 


EAM384
r-miR-333_rfam7.0
/5AmMC6/AAAAGTAACTAGCACACCAC
(SEQ. ID. No. 591)


 


EAM385
hmr-miR-335_rfam7.0
/5AmMC6/ACATTTTTCGTTATTGCTCTT
(SEQ. ID. No. 592)


 


EAM393
r-miR-7*_rfam7.0
/5AmMC6/TATGGCAGACTGTGATTTGTTG
(SEQ. ID. No. 593)


 


EAM304
hmr-miR-200a_rfam7.0
/5AmMC6/CATCGTTACCAGACAGTGTTA
(SEQ. ID. No. 594)


 


EAM298
hmr-miR-194_rfam7.0
/5AmMC6/TCCACATGGAGTTGCTGTTACA
(SEQ. ID. No. 595)


 


JLA221
hmr-miR-191_rfam7.0
/5AmMC6/AACAGCTGCTTTTGGGATTCTG
(SEQ. ID. No. 596)


 


EAM295
hmr-miR-190_rfam7.0
/5AmMC6/ACCTAATATATCAAACATATCA
(SEQ. ID. No. 597)


 


EAM292
hmr-miR-186_rfam7.0
/5AmMC6/AAGCCCAAAAGGAGAATTCTTTG
(SEQ. ID. No. 598)


 


JLA219
hmr-miR-185_rfam7.0
/5AmMC6/AGGAACTGCCTTTCTCTCCAA
(SEQ. ID. No. 599)


 


EAM290
hmr-miR-184_rfam7.0
/5AmMC6/ACCCTTATCAGTTCTCCGTCCA
(SEQ. ID. No. 600)


 


EAM402
hm-miR-133b_rfam7.0
/5AmMC6/TAGCTGGTTGAAGGGGACCAA
(SEQ. ID. No. 601)


 


EAM403
h-miR-151_rfam7.0
/5AmMC6/CCTCAAGGAGCTTCAGTCTAGT
(SEQ. ID. No. 602)


 


EAM404
hmr-miR-196b_rfam7.0
/5AmMC6/CCAACAACAGGAAACTACCTA
(SEQ. ID. No. 603)


 


EAM418
hm-miR-370_rfam7.0
/5AmMC6/CCAGGTTCCACCCCAGCAGG
(SEQ. ID. No. 604)


 


EAM419
h-miR-371 rfam7.0
/5AmMC6/ACACTCAAAAGATGGCGGCA
(SEQ. ID. No. 605)


 


EAM420
h-miR-372_rfam7.0
/5AmMC6/ACGCTCAAATGTCGCAGCAC
(SEQ. ID. No. 606)


 


EAM421
h-miR-373_rfam7.0
/5AmMC6/ACACCCCAAAATCGAAGCAC
(SEQ. ID. No. 607)


 


EAM422
h-miR-373*_rfam7.0
/5AmMC6/GGAAAGCGCCCCCATTTTGA
(SEQ. ID. No. 608)


 


EAM423
h-miR-374_rfam7.0
/5AmMC6/CACTTATCAGGTTGTATTATAA
(SEQ. ID. No. 609)


 


EAM426
m-miR-215 rfam7.0
/5AmMC6/GTCTGTCAAATCATAGGTCAT
(SEQ. ID. No. 610)


 


EAM427
hm-miR-409-3p_rfam7.0
/5AmMC6/GGGGTTCACCGAGCAACATTC
(SEQ. ID. No. 611)


 


EAM428
hm-miR-410_rfam7.0
/5AmMC6/CAGGCCATCTGTGTTATATT
(SEQ. ID. No. 612)


 


EAM429
m-miR-376b_rfam7.0
/5AmMC6/AGTGGATGTTCCTCTATGAT
(SEQ. ID. No. 613)


 


EAM430
m-miR-376a_rfam7.0
/5AmMC6/CGTGGATTTTCCTCTACGAT
(SEQ. ID. No. 614)


 


EAM431
m-miR-411_rfam7.0
/5AmMC6/GAGGGTTAGTGGACCGTGTT
(SEQ. ID. No. 615)


 


EAM432
m-miR-380-3p_rfam7.0
/5AmMC6/GATGTGGACCATACTACATA
(SEQ. ID. No. 616)


 


EAM433
hm-miR-412_rfam7.0
/5AmMC6/GGCTAGTGGACCAGGTGAAG
(SEQ. ID. No. 617)


 


EAM264
hmr-miR-27b_rfam7.0
/5AmMC6/CAGAACTTAGCCACTGTGAA
(SEQ. ID. No. 618)


 


EAM263
hmr-miR-26a_rfam7.0
/5AmMC6/AGCCTATCCTGGATTACTTGAA
(SEQ. ID. No. 619)


 


EAM262
hmr-miR-24_rfam7.0
/5AmMC6/CTGTTCCTGCTGAACTGAGCCA
(SEQ. ID. No. 620)


 


EAM261
hmr-miR-23b_rfam7.0
/5AmMC6/GTGGTAATCCCTGGCAATGTGAT
(SEQ. ID. No. 621)


 


EAM260
hmr-miR-23a_rfam7.0
/5AmMC6/GGAAATCCCTGGCAATGTGAT
(SEQ. ID. No. 622)


 


EAM256
h-miR-220_rfam7.0
/5AmMC6/AAAGTGTCAGATACGGTGTGG
(SEQ. ID. No. 623)


 


EAM255
hmr-miR-22_rfam7.0
/5AmMC6/ACAGTTCTTCAACTGGCAGCTT
(SEQ. ID. No. 624)


 


EAM248
hmr-miR-213_rfam7.0
/5AmMC6/GGTACAATCAACGGTCGATGGT
(SEQ. ID. No. 625)


 


EAM244
hmr-miR-21_rfam7.0
/5AmMC6/TCAACATCAGTCTGATAAGCTA
(SEQ. ID. No. 626)


 


EAM240
hmr-miR-20a_rfam7.0
/5AmMC6/CTACCTGCACTATAAGCACTTTA
(SEQ. ID. No. 627)


 


EAM237
hmr-miR-19b_rfam7.0
/5AmMC6/TCAGTTTTGCATGGATTTGCACA
(SEQ. ID. No. 628)


 


EAM233
hmr-miR-196a rfam7.0
/5AmMC6/CCCAACAACATGAAACTACCTA
(SEQ. ID. No. 629)


 


EAM214
hm-miR-148a_rfam7.0
/5AmMC6/ACAAAGTTCTGTAGTGCACTGA
(SEQ. ID. No. 630)


 


EAM212
hmr-miR-145_rfam7.0
/5AmMC6/AAGGGATTCCTGGGAAAACTGGAC
(SEQ. ID. No. 631)


 


EAM211
hmr-miR-144_rfam7.0
/5AmMC6/CTAGTACATCATCTATACTGTA
(SEQ. ID. No. 632)


 


EAM210
hmr-miR-143_rfam7.0
/5AmMC6/tgAGCTACAGTGCTTCATCTCA
(SEQ. ID. No. 633)


 


EAM389
r-miR-346 rfam7.0
/5AmMC6/AGAGGCAGGCACTCAGGCAGA
(SEQ. ID. No. 634)


 


EAM390
r-miR-347_rfam7.0
/5AmMC6/TGGGCGACCCAGAGGGACA
(SEQ. ID. No. 635)


 


EAM391
r-miR-349_rfam7.0
/5AmMC6/AGAGGTTAAGACAGCAGGGCTG
(SEQ. ID. No. 636)


 


JLA223
hmr-miR-33_rfam7.0
/5AmMC6/TGCAATGCAACTACAATGCACC
(SEQ. ID. No. 637)


 


EAM277
hmr-miR-96_rfam7.0
/5AmMC6/GCAAAAATGTGCTAGTGCCAAA
(SEQ. ID. No. 638)


 


EAM276
hmr-miR-9_rfam7.0
/5AmMC6/TCATACAGCTAGATAACCAAAGA
(SEQ. ID. No. 639)


 


EAM272
hmr-miR-30d_rfam7.0
/5AmMC6/CTTCCAGTCGGGGATGTTTACA
(SEQ. ID. No. 640)


 


EAM288
mr-miR-10b_rfam7.0
/5AmMC6/ACACAAATTCGGTTCTACAGGG
(SEQ. ID. No. 641)


 


EAM293
hm-miR-188_rfam7.0
/5AmMC6/ACCCTCCACCATGCAAGGGATG
(SEQ. ID. No. 642)


 


EAM297
hmr-miR-193a_rfam7.0
/5AmMC6/CTGGGACTTTGTAGGCCAGTT
(SEQ. ID. No. 643)


 


EAM301
h-miR-198_rfam7.0
/5AmMC6/CCTATCTCCCCTCTGGACC
(SEQ. ID. No. 644)


 


EAM232
hmr-miR-192_rfam7.0
/5AmMC6/GGCTGTCAATTCATAGGTCAG
(SEQ. ID. No. 645)


 


EAM231
hmr-miR-187_rfam7.0
/5AmMC6/CGGCTGCAACACAAGACACGA
(SEQ. ID. No. 646)


 


EAM230
hmr-miR-183_rfam7.0
/5AmMC6/CAGTGAATTCTACCAGTGCCATA
(SEQ. ID. No. 647)


 


EAM229
hm-miR-182_rfam7.0
/5AmMC6/TGTGAGTTCTACCATTGCCAAA
(SEQ. ID. No. 648)


 


EAM220
hmr-miR-154_rfam7.0
/5AmMC6/CGAAGGCAACACGGATAACCTA
(SEQ. ID. No. 649)


 


EAM219
hmr-miR-153_rfam7.0
/5AmMC6/TCACTTTTGTGACTATGCAA
(SEQ. ID. No. 650)


 


EAM218
hmr-miR-152_rfam7.0
/5AmMC6/CCAAGTTCTGTCATGCACTGA
(SEQ. ID. No. 651)


 


EAM217
hmr-miR-150_rfam7.0
/5AmMC6/ACACTGGTACAAGGGTTGGGAGA
(SEQ. ID. No. 652)


 


EAM216
hm-miR-149_rfam7.0
/5AmMC6/GGAGTGAAGACACGGAGCCAGA
(SEQ. ID. No. 653)


 


EAM215
hmr-miR-148b_rfam7.0
/5AmMC6/ACAAAGTTCTGTGATGCACTGA
(SEQ. ID. No. 654)


 


EAM271
hmr-miR-30c rfam7.0
/5AmMC6/GCTGAGAGTGTAGGATGTTTACA
(SEQ. ID. No. 655)


 


EAM268
hmr-miR-29a_rfam7.0
/5AmMC6/AACCGATTTCAGATGGTGCTAG
(SEQ. ID. No. 656)


 


EAM305
hmr-miR-200b_rfam7.0
/5AmMC6/GTCATCATTACCAGGCAGTATTA
(SEQ. ID. No. 657)


 


EAM303
hm-miR-199a*_rfam7.0
/5AmMC6/AACCAATGTGCAGACTACTGTA
(SEQ. ID. No. 658)


 


EAM300
h-miR-197_rfam7.0
/5AmMC6/GCTGGGTGGAGAAGGTGGTGAA
(SEQ. ID. No. 659)


 


EAM299
hmr-miR-195 rfam7.0
/5AmMC6/GCCAATATTTCTGTGCTGCTA
(SEQ. ID. No. 660)


 


JLA91
hmr-miR-99b_rfam7.0
/5AmMC6/CGCAAGGTCGGTTCTACGGGTG
(SEQ. ID. No. 661)


 


JLA92
hmr-miR-433_rfam7.0
/5AmMC6/ACACCGAGGAGCCCATCATGAT
(SEQ. ID. No. 662)


 


JLA93
hmr-miR-431_rfam7.0
/5AmMC6/CCTGCATGACGGCCTGCAAGACA
(SEQ. ID. No. 663)


 


JLA94
hmr-miR-365_rfam7.0
/5AmMC6/ATAAGGATTTTTAGGGGCATTA
(SEQ. ID. No. 664)


 


JLA95
hmr-miR-450_rfam7.0
/5AmMC6/TATTAGGAACACATCGCAAAAA
(SEQ. ID. No. 665)


 


JLA96
hmr-miR-449_rfam7.0
/5AmMC6/ACCAGCTAACAATACACTGCCA
(SEQ. ID. No. 666)


 


JLA99
hmr-miR-448_rfam7.0
/5AmMC6/ATGGGACATCCTACATATGCAA
(SEQ. ID. No. 667)


 


JLA103
hmr-miR-424_rfam7.0
/5AmMC6/TTCAAAACATGAATTGCTGCTG
(SEQ. ID. No. 668)


 


JLA105
hm-miR-361_rfam7.0
/5AmMC6/GTACCCCTGGAGATTCTGATAA
(SEQ. ID. No. 669)


 


JLA106
hm-miR-375_rfam7.0
/5AmMC6/TCACGCGAGCCGAACGAACAAA
(SEQ. ID. No. 670)


 


JLA107
hm-miR-377_rfam7.0
/5AmMC6/ACAAAAGTTGCCTTTGTGTGAT
(SEQ. ID. No. 671)


 


JLA108
hm-miR-378_rfam7.0
/5AmMC6/ACACAGGACCTGGAGTCAGGAG
(SEQ. ID. No. 672)


 


JLA109
hm-miR-379_rfam7.0
/5AmMC6/CCTACGTTCCATAGTCTACCA
(SEQ. ID. No. 673)


 


JLA110
hm-miR-380-5p_rfam7.0
/5AmMC6/GCGCATGTTCTATGGTCAACCA
(SEQ. ID. No. 674)


 


JLA111
hm-miR-381_rfam7.0
/5AmMC6/ACAGAGAGCTTGCCCTTGTATA
(SEQ. ID. No. 675)


 


JLA112
hm-miR-382_rfam7.0
/5AmMC6/CGAATCCACCACGAACAACTTC
(SEQ. ID. No. 676)


 


JLA115
hm-miR-384_rfam7.0
/5AmMC6/TATGAACAATTTCTAGGAAT
(SEQ. ID. No. 677)


 


JLA116
hm-miR-425_rfam7.0
/5AmMC6/GGCGGACACGACATTCCCGAT
(SEQ. ID. No. 678)


 


JLA117
hm-miR-452 rfam7.0
/5AmMC6/GTCTCAGTTTCCTCTGCAAACA
(SEQ. ID. No. 679)


 


JLA118
hm-miR-30e-3p_rfam7.0
/5AmMC6/GCTGTAAACATCCGACTGAAAG
(SEQ. ID. No. 680)


 


JLA104
mr-miR-129-3p_rfam7.0
/5AmMC6/ATGCTTTTTGGGGTAAGGGCTT
(SEQ. ID. No. 681)


 


JLA98
mr-miR-429_rfam7.0
/5AmMC6/ACGGCATTACCAGACAGTATTA
(SEQ. ID. No. 682)


 


JLA101
mr-miR-330_rfam7.0
/5AmMC6/TCTCTGCAGGCCCTGTGCTTTGC
(SEQ. ID. No. 683)


 


JLA102
mr-miR-322 rfam7.0
/5AmMC6/TGTTGCAGCGCTTCATGTTT
(SEQ. ID. No. 684)


 


JLA114
m-miR-383_rfam7.0
/5AmMC6/AGCCACAGTCACCTTCTGATCT
(SEQ. ID. No. 685)


 


JLA5
hmr-miR-451
/5AmMC6/AAACTCAGTAATGGTAACGGTTT
(SEQ. ID. No. 686)


 


JLA201
r-miR-421_rfam7.0
/5AmMC6/CAACAAACATTTAATGAGGCC
(SEQ. ID. No. 687)


 


JLA202
m-miR-463_rfam7.0
/5AmMC6/TGATGGACAACAAATTAGGTA
(SEQ. ID. No. 688)


 


JLA203
m-miR-464_rfam7.0
/5AmMC6/TATCTCACAGAATAAACTTGGTA
(SEQ. ID. No. 689)


 


JLA204
m-miR-465_rfam7.0
/5AmMC6/TCACATCAGTGCCATTCTAAATA
(SEQ. ID. No. 690)


 


JLA205
m-miR-466_rfam7.0
/5AmMC6/GTCTTATGTGTGCGTGTATGTAT
(SEQ. ID. No. 691)


 


JLA206
m-miR-467_rfam7.0
/5AmMC6/GTGTAGGTGTGTGTATGTATAT
(SEQ. ID. No. 692)


 


JLA207
m-miR-468_rfam7.0
/5AmMC6/CAGACACACGCACATCAGTCATA
(SEQ. ID. No. 693)


 


JLA208
m-miR-469_rfam7.0
/5AmMC6/GGACACCAAGATCAATGAAAGAGGCA
(SEQ. ID. No. 694)


 


JLA209
m-miR-470_rfam7.0
/5AmMC6/TCACCAGTGCCAGTCCAAGAA
(SEQ. ID. No. 695)


 


JLA210
m-miR-471_rfam7.0
/5AmMC6/TGTGAAAAGCACTATACTACGTA
(SEQ. ID. No. 696)


 


EAM325
hmr-miR-27a_rfam7.0
/5AmMC6/GGCGGAACTTAGCCACTGTGAA
(SEQ. ID. No. 697)


 


EAM326
hmr-miR-296_rfam7.0
/5AmMC6/ACAGGATTGAGGGGGGGCCCT
(SEQ. ID. No. 698)


 


EAM327
hmr-miR-299-5p_rfam7.0
/5AmMC6/ATGTATGTGGGACGGTAAACCA
(SEQ. ID. No. 699)


 


EAM328
hmr-miR-301_rfam7.0
/5AmMC6/GCTTTGACAATACTATTGCACTG
(SEQ. ID. No. 700)


 


EAM329
hm-miR-302a_rfam7.0
/5AmMC6/TCACCAAAACATGGAAGCACTTA
(SEQ. ID. No. 701)


 


EAM330
hmr-miR-30a-5p_rfam7.0
/5AmMC6/GCTTCCAGTCGAGGATGTTTACA
(SEQ. ID. No. 702)


 


EAM331
hmr-miR-30e-5p_rfam7.0
/5AmMC6/TCCAGTCAAGGATGTTTACA
(SEQ. ID. No. 703)


 


EAM332
hmr-miR-31_rfam7.0
/5AmMC6/CAGCTATGCCAGCATCTTGCCT
(SEQ. ID. No. 704)


 


EAM333
hmr-miR-32 rfam7.0
/5AmMC6/GCAACTTAGTAATGTGCAATA
(SEQ. ID. No. 705)


 


EAM335
h-miR-34b_rfam7.0
/5AmMC6/CAATCAGCTAATGACACTGCCT
(SEQ. ID. No. 706)


 


EAM336
hmr-miR-34c_rfam7.0
/5AmMC6/GCAATCAGCTAACTACACTGCCT
(SEQ. ID. No. 707)


 


EAM337
hmr-miR-93_rfam7.0
/5AmMC6/CTACCTGCACGAACAGCACTTTG
(SEQ. ID. No. 708)


 


EAM208
hmr-miR-141_rfam7.0
/5AmMC6/CCATCTTTACCAGACAGTGTT
(SEQ. ID. No. 709)


 


EAM207
hmr-miR-140 rfam7.0
/5AmMC6/CTACCATAGGGTAAAACCACT
(SEQ. ID. No. 710)


 


JLA222
hmr-miR-139_rfam7.0
/5AmMC6/TGGAGACACGTGCACTGTAGA
(SEQ. ID. No. 711)


 


JLA220
hmr-miR-138_rfam7.0
/5AmMC6/CCTGATTCACAACACCAGCTG
(SEQ. ID. No. 712)


 


EAM203
hmr-miR-135a_rfam7.0
/5AmMC6/TTCACATAGGAATAAAAAGCCATA
(SEQ. ID. No. 713)


 


EAM200
hmr-miR-133a_rfam7.0
/5AmMC6/ACAGCTGGTTGAAGGGGACCAA
(SEQ. ID. No. 714)


 


EAM195
hmr-miR-128b_rfam7.0
/5AmMC6/GAAAGAGACCGGTTCACTGTGA
(SEQ. ID. No. 715)


 


EAM194
hmr-miR-128a_rfam7.0
/5AmMC6/AAAAGAGACCGGTTCACTGTGA
(SEQ. ID. No. 716)


 


EAM254
hmr-miR-219_rfam7.0
/5AmMC6/AGAATTGCGTTTGGACAATCA
(SEQ. ID. No. 717)


 


EAM257
hmr-miR-221_rfam7.0
/5AmMC6/GAAACCCAGCAGACAATGTAGCT
(SEQ. ID. No. 718)


 


EAM258
hmr-miR-222_rfam7.0
/5AmMC6/GAGACCCAGTAGCCAGATGTAGCT
(SEQ. ID. No. 719)


 


EAM259
hmr-miR-223_rfam7.0
/5AmMC6/GGGGTATTTGACAAACTGACA
(SEQ. ID. No. 720)


 


JLA211
m-miR-434-5p_rfam7.0
/5AmMC6/GGTTCAAACCATGAGTCGAGCT
(SEQ. ID. No. 721)


 


JLA212
m-miR-434-3p_rfam7.0
/5AmMC6/GGAGTCGAGTGATGGTTCAAA
(SEQ. ID. No. 722)


 


JLA213
m-miR-433-5p_rfam7.0
/5AmMC6/GAATAATGACAGGCTCACCGTA
(SEQ. ID. No. 723)


 


JLA2
hsa-miR-522
/5AmMC6/ACACTCTAAAGGGAACCATTTT
(SEQ. ID. No. 724)


 


JLA3
hsa-miR-495
/5AmMC6/AAAGAAGTGCACCATGTTTGTTT
(SEQ. ID. No. 725)


 


JLA200
r-miR-297_rfam7.0
/5AmMC6/CATGCATACATGCACACATACAT
(SEQ. ID. No. 726)


 


JLA6
hsa-miR-518e
/5AmMC6/AACACTCTGAAGGGAAGCGC
(SEQ. ID. No. 727)


 


JLA7
hsa-miR-519a
/5AmMC6/CACTCTAAAAGGATGCACTTT
(SEQ. ID. No. 728)


 


JLA8
hsa-mir-527*
/5AmMC6/TTCACCAAAGGGAAGCACTTT
(SEQ. ID. No. 729)


 


JLA72
hmr-miR-140*_rfam7.0
/5AmMC6/GTCCGTGGTTCTACCCTGTGG
(SEQ. ID. No. 730)


 


JLA10
hsa-miR-521
/5AmMC6/ACACTCTAAAGGGAAGTGCGTT
(SEQ. ID. No. 731)


 


JLA12
hsa-miR-362
/5AmMC6/CTCACACCTAGGTTCCAAGGATT
(SEQ. ID. No. 732)


 


JLA74
hsa-mir-18*
/5AmMC6/CCAGAAGGAGCACTTAGGGCAG
(SEQ. ID. No. 733)


 


JLA14
hm-miR-363
/5AmMC6/TTACAGATGGATACCGTGCAATT
(SEQ. ID. No. 734)


 


JLA77
hsa-mir-19b-1*
/5AmMC6/GCTGGATGCAAACCTGCAAAAC
(SEQ. ID. No. 735)


 


JLA17
hsa-mir-520c, b, f
/5AmMC6/CCTCTAAAAGGAAGCACTTTCT
(SEQ. ID. No. 736)


 


JLA79
hsa-mir-23a*
/5AmMC6/AATCCCATCCCCAGGAACCC
(SEQ. ID. No. 737)


 


JLA20
hsa-miR-369-5p
/5AmMC6/CGAATATAACACGGTCGATCT
(SEQ. ID. No. 738)


 


JLA81
hsa-mir-339*
/5AmMC6/CGGCTCTGTCGTCGAGGCGC
(SEQ. ID. No. 739)


 


JLA23
hsa-mir-342*
/5AmMC6/TCAATCACAGATAGCACCCCT
(SEQ. ID. No. 740)


 


JLA24
hsa-mir-19a*
/5AmMC6/GTAGTGCAACTATGCAAAACT
(SEQ. ID. No. 741)


 


JLA26
hsa-miR-517a, b
/5AmMC6/ACACTCTAAAGGGATGCACGAT
(SEQ. ID. No. 742)


 


JLA27
hsa-miR-516-5p
/5AmMC6/AAAGTGCTTCTTACCTCCAGAT
(SEQ. ID. No. 743)


 


JLA28
hsa-miR-518b
/5AmMC6/ACCTCTAAAGGGGAGCGCTT
(SEQ. ID. No. 744)


 


JLA29
hsa-miR-519d
/5AmMC6/ACACTCTAAAGGGAGGCACTTT
(SEQ. ID. No. 745)


 


JLA73
hr-mir-151*
/5AmMC6/ATACTAGACTGTGAGCTCCTCGA
(SEQ. ID. No. 746)


 


JLA31
hsa-mir-28*
/5AmMC6/TCCAGGAGCTCACAATCTAGTG
(SEQ. ID. No. 747)


 


JLA33
hsa-mir-519a-2*
/5AmMC6/AGAAAGCGCTTCCCTGTAGAG
(SEQ. ID. No. 748)


 


JLA34
hsa-mir-26b*
/5AmMC6/AGCCAAGTAATGGAGAACAGG
(SEQ. ID. No. 749)


 


JLA35
hsa-miR-526c
/5AmMC6/AGAAAGCGCTTCCCTCTAGAG
(SEQ. ID. No. 750)


 


JLA36
hsa-miR-527
/5AmMC6/ACAGAAAGGGCTTCCCTTTGC
(SEQ. ID. No. 751)


 


JLA38
hsa-mir-29b-2*
/5AmMC6/TCTAAGCCACCATGTGAAACCA
(SEQ. ID. No. 752)


 


JLA39
hsa-let-7g*
/5AmMC6/GCAAGGCAGTGGCCTGTACA
(SEQ. ID. No. 753)


 


JLA40
hsa-miR-518a
/5AmMC6/TCCAGCAAAGGGAAGCGCTT
(SEQ. ID. No. 754)


 


JLA41
hsa-mi R-523
/5AmMC6/ACCCTCTATAGGGAAGCGCGT
(SEQ. ID. No. 755)


 


JLA44
hsa-miR-515-3p
/5AmMC6/AACGCTCCAAAAGAAGGCACT
(SEQ. ID. No. 756)


 


JLA45
hsa-mir-146b*
/5AmMC6/ACCAGAACTGAGTCCACAGGG
(SEQ. ID. No. 757)


 


JLA49
hsa-mir-222*
/5AmMC6/ATCTACACTGGCTACTGAGCC
(SEQ. ID. No. 758)


 


JLA53
hsa-mir-24*
/5AmMC6/ACTGTGTTTCAGCTCAGTAGGCA
(SEQ. ID. No. 759)


 


JLA55
hsa-miR-503
/5AmMC6/ACTGCAGAACTGTTCCCGCTG
(SEQ. ID. No. 760)


 


JLA57
hsa-mir-505
/5AmMC6/AGAGGAAACCAGCAAGTGTTGA
(SEQ. ID. No. 761)


 


JLA82
hsa-mir-423*
/5AmMC6/AAAGTCTCGCTCTCTGCCCCT
(SEQ. ID. No. 762)


 


JLA66
hsa-miR-432
/5AmMC6/CCACCCAATGACCTACTCCAAG
(SEQ. ID. No. 763)


 


JLA83
hsa-mir-425*
/5AmMC6/TCAACGGGAGTGATCGTGTCAT
(SEQ. ID. No. 764)


 


JLA84
hsa-mir-92-1*
/5AmMC6/AGCATTGCAACCGATCCCAAC
(SEQ. ID. No. 765)


 


JLA69
hsa-mir-193*
/5AmMC6/TCATCTCGCCCGCAAAGACC
(SEQ. ID. No. 766)


 


JLA70
hsa-miR-515-5p
/5AmMC6/AGAAAGTGCTTTCTTTTGGAGAA
(SEQ. ID. No. 767)


 


JLA71
hsa-mir-516-1*
/5AmMC6/GAAAGTGCTTCTTTCCTCGAGAA
(SEQ. ID. No. 768)


 


JLA85
hsa-mir-30d*
/5AmMC6/GCAGCAAACATCTGACTGAAAG
(SEQ. ID. No. 769)


 


JLA125
h-miR-20b_rfam7.0
/5AmMC6/CTACCTGCACTATGAGCACTTTG
(SEQ. ID. No. 770)


 


JLA198
h-miR-191*_rfam7.0
/5AmMC6/GGGGACGAAATCCAAGCGCAGC
(SEQ. ID. No. 771)


 


JLA199
h-miR-154*_rfam7.0
/5AmMC6/AATAGGTCAACCGTGTATGATT
(SEQ. ID. No. 772)


 


EAM316
h-miR-147_rfam7.0
/5AmMC6/GCAGAAGCATTTCCACACAC
(SEQ. ID. No. 773)


 


EAM317
h-miR-155_rfam7.0
/5AmMC6/CCCCTATCACGATTAGCATTAA
(SEQ. ID. No. 774)


 


EAM318
h-miR-17-3p_rfam7.0
/5AmMC6/ACAAGTGCCTTCACTGCAGT
(SEQ. ID. No. 775)


 


JLA195
h-miR-200a*_rfam7.0
/5AmMC6/TCCAGCACTGTCCGGTAAGATG
(SEQ. ID. No. 776)


 


JLA196
h-miR-302a*_rfam7.0
/5AmMC6/AAAGCAAGTACATCCACGTTTA
(SEQ. ID. No. 777)


 


JLA197
h-miR-299-3p_rfam7.0
/5AmMC6/AAGCGGTTTACCATCCCACATA
(SEQ. ID. No. 778)


 


EAM319
h-miR-182* rfam7.0
/5AmMC6/TAGTTGGCAAGTCTAGAACCA
(SEQ. ID. No. 779)


 


EAM405
h-miR-302b_rfam7.0
/5AmMC6/CTACTAAAACATGGAAGCACTTA
(SEQ. ID. No. 780)


 


EAM406
h-miR-302b*_rfam7.0
/5AmMC6/AGAAAGCACTTCCATGTTAAAGT
(SEQ. ID. No. 781)


 


EAM392
r-miR-352_rfam7.0
/5AmMC6/TACTATGCAACCTACTACTCT
(SEQ. ID. No. 782)


 


JLA123
h-miR-423_rfam7.0
/5AmMC6/CTGAGGGGCCTCAGACCGAGCT
(SEQ. ID. No. 783)


 


JLA124
h-miR-18b_rfam7.0
/5AmMC6/TAACTGCACTAGATGCACCTTA
(SEQ. ID. No. 784)
















TABLE 6





Normalized miRNA expression profiling data for MEP, ERY and MEGA samples



























ProbeID
Description
MEP_1
MEP_2
MEP_3
MEP_4
MEP_5
MEP_6
MEP_7
MEP_8
ERY1_1






EAM190
h-miR-10b
6.00
6.78
6.00
6.00
6.44
6.00
6.06
6.00
6.00



EAM187
hmr-miR-107
7.17
6.00
6.65
6.88
6.00
7.44
6.00
6.97
6.85



EAM185
hmr-miR-103
7.59
6.00
7.28
7.38
6.00
7.94
6.00
7.60
7.26



EAM181
hmr-let-7f
9.18
9.82
10.08
10.11
9.68
9.80
9.61
8.96
9.40



EAM179
hmr-let-7d
7.47
9.56
8.99
9.45
8.89
9.76
9.10
9.03
8.76



EAM177
mr-miR-101b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM175
hmr-miR-320
9.41
8.52
8.86
9.14
9.42
9.49
9.67
9.85
9.17



EAM168
hmr-let-7e
6.00
6.39
6.35
6.00
6.00
6.00
6.00
6.00
6.19



EAM161
hmr-miR-28
6.00
6.00
6.00
6.00
6.00
6.00
7.60
6.00
6.00



EAM160
hmr-miR-26b
8.92
8.03
9.08
9.13
8.76
8.24
8.82
7.56
8.36



EAM155
hmr-miR-136
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM283
mr-miR-211
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM282
m-miR-199b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM281
mr-miR-217
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM280
hmr-miR-30a-
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00




3p












EAM279
hmr-miR-29c
6.00
6.98
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM278
hmr-miR-98
6.00
7.19
7.59
6.00
7.17
6.00
6.00
6.00
6.74



EAM270
hmr-miR-30b
7.89
7.61
8.70
9.14
8.89
9.10
9.01
9.49
8.81



EAM159
hmr-miR-130a
7.76
8.35
8.79
8.49
8.39
8.29
8.96
9.53
8.41



EAM163
hmr-miR-142-
6.00
6.00
6.00
6.42
6.00
7.29
6.77
6.00
6.39




3p












EAM171
hmr-miR-137
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM306
m-miR-201
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM307
m-miR-202
6.00
6.00
6.00
6.00
6.42
6.00
6.00
6.00
6.00



EAM308
hmr-miR-206
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM309
m-miR-207
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM310
hmr-miR-208
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM247
hmr-miR-212
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM251
hmr-miR-216
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM253
hmr-miR-218
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM275
hmr-miR-34a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM246
h-miR-211
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM250
h-miR-215
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM252
h-miR-217
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM224
hmr-miR-17-5p
11.37
11.51
11.39
10.79
11.11
11.33
11.35
11.44
11.52



EAM225
hmr-miR-18a
6.00
6.00
6.00
7.67
6.07
6.00
6.00
8.32
8.14



EAM226
hmr-miR-181a
6.26
8.16
7.40
8.99
9.49
8.48
8.90
7.90
8.54



EAM227
hmr-miR-181b
6.00
6.00
8.18
8.18
6.59
7.95
7.59
9.07
7.76



EAM234
hmr-miR-199a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM235
h-miR-199b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM236
hmr-miR-19a
7.89
7.04
8.46
8.23
8.32
7.24
7.94
7.49
8.65



EAM241
hmr-miR-203
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM242
hmr-miR-204
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM243
hmr-miR-205
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM245
hmr-miR-210
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM249
hmr-miR-214
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM184
hmr-miR-100
6.00
6.00
6.26
6.00
6.00
6.00
6.00
6.00
6.00



EAM186
h-miR-106a
11.10
11.31
11.28
10.79
10.90
11.00
11.02
11.32
11.22



EAM189
hmr-miR-10a
8.48
7.98
7.43
7.74
7.44
6.44
7.37
6.00
7.48



EAM191
hmr-miR-122a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM192
hmr-miR-126*
7.32
6.00
7.66
6.20
6.62
6.00
6.76
7.63
7.39



EAM198
hmr-miR-130b
6.00
6.00
6.36
6.44
6.68
6.00
6.00
6.68
6.00



EAM202
hmr-miR-134
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM209
hmr-miR-142-
6.00
7.72
7.42
6.95
8.16
6.62
7.29
7.92
6.00




5p












EAM221
m-miR-155
7.57
8.58
7.87
7.76
7.43
7.79
7.65
7.47
7.97



EAM223
hmr-miR-15b
10.67
8.82
10.83
10.79
10.29
10.53
10.47
9.94
10.59



EAM228
hmr-miR-181c
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM222
hm-miR-15a
8.94
6.00
8.11
9.87
8.82
8.92
8.20
8.39
8.30



EAM111
hm-let-7g
10.22
9.62
10.09
10.09
9.76
10.06
9.73
9.61
9.34



EAM131
hmr-miR-92
11.06
11.53
11.55
10.98
11.69
11.74
11.75
11.88
11.30



EAM139
hmr-miR-146a
9.03
9.70
6.00
9.07
9.31
6.00
8.53
9.49
9.22



EAM145
hmr-let-7c
9.28
9.27
9.03
9.67
9.39
9.04
9.24
9.14
8.71



EAM109
hmr-miR-7
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM152
hm-miR-9*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA215
hmr-let-7i
9.03
8.00
8.07
8.44
7.26
8.38
8.85
7.71
9.25



EAM153
hmr-let-7a
11.22
10.91
10.80
10.07
11.01
10.77
11.09
10.93
10.63



EAM147
hmr-let-7b
9.07
6.31
9.13
8.64
9.00
9.63
10.04
9.43
7.66



EAM137
hmr-miR-132
6.00
6.00
6.00
6.00
6.34
6.00
6.00
6.00
6.00



EAM133
hmr-miR-324-
7.74
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00




5p












EAM103
hmr-miR-124a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM105
hmr-miR-125b
6.00
7.69
7.56
8.05
8.75
8.32
6.81
7.30
8.45



EAM121
hmr-miR-99a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM115
hmr-miR-16
12.32
12.42
12.31
11.40
11.87
12.30
11.83
11.81
12.08



EAM119
hmr-miR-29b
6.00
6.00
6.00
6.93
6.00
6.00
6.00
6.00
6.00



EAM311
hmr-miR-101
6.00
6.00
6.00
6.00
6.00
6.00
6.39
6.00
6.00



EAM312
h-miR-105
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM313
hmr-miR-106b
9.31
9.44
8.80
9.39
9.18
9.09
8.34
8.86
8.75



EAM314
hmr-miR-126
8.75
9.89
8.72
10.12
9.41
8.10
9.63
8.69
10.57



EAM315
hmr-miR-127
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM320
hm-miR-189
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA216
hmr-miR-200c
6.00
6.00
6.00
6.00
6.00
6.00
8.00
6.00
7.19



EAM323
h-miR-224
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM324
hmr-miR-25
6.00
9.01
6.41
7.40
8.30
7.82
8.26
8.59
7.82



EAM386
r-miR-336
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA218
r-miR-343
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM388
r-miR-344
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM338
h-miR-95
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA214
hmr-miR-129
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM340
mr-let-7d*
7.71
6.00
6.00
6.00
6.00
7.41
6.00
6.00
6.00



EAM341
m-miR-106a
10.34
11.12
10.40
9.97
10.46
10.70
10.47
10.68
10.60



EAM342
hmr-miR-135b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM343
mr-miR-151
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM344
m-miR-17-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM345
m-miR-224
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM346
mr-miR-290
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM347
mr-miR-291-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM348
mr-miR-291-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM349
mr-miR-292-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM350
mr-miR-292-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM351
m-miR-293
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM352
m-miR-294
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM353
m-miR-295
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM354
m-miR-297
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM355
mr-miR-298
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM356
mr-miR-300
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM358
hmr-miR-323
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM359
hmr-miR-324-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
7.59
6.00



EAM360
mr-miR-325
7.36
6.93
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM361
hmr-miR-326
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM362
hmr-miR-328
6.00
6.00
6.98
6.00
6.00
6.00
6.00
6.00
6.00



EAM363
mr-miR-329
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM365
hmr-miR-331
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM366
mr-miR-337
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM367
hmr-miR-338
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM368
hmr-miR-339
6.00
6.00
6.00
6.00
6.10
6.00
6.00
6.00
6.00



EAM369
hmr-miR-340
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM370
mr-miR-341
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM371
hmr-miR-342
9.66
9.70
6.00
10.47
9.48
9.58
10.06
9.41
8.70



EAM372
m-miR-344
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM373
mr-miR-345
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM374
m-miR-346
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM375
mr-miR-34b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA217
mr-miR-350
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM377
mr-miR-351
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM378
mr-miR-7b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM382
r-miR-20*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM383
r-miR-327
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM384
r-miR-333
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM385
hmr-miR-335
6.91
7.60
8.50
7.99
7.28
6.00
7.20
7.07
7.65



EAM393
r-miR-7*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM304
hmr-miR-200a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM298
hmr-miR-194
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA221
hmr-miR-191
7.79
6.44
7.74
8.72
8.73
8.28
8.34
8.43
7.85



EAM295
hmr-miR-190
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM292
hmr-miR-186
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA219
hmr-miR-185
6.00
6.00
6.00
6.07
7.36
7.59
6.40
6.00
6.00



EAM290
hmr-miR-184
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM402
hm-miR-133b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM403
h-miR-151
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM404
hmr-miR-196b
6.00
6.00
6.00
6.52
6.00
6.00
6.00
6.00
6.00



EAM418
hm-miR-370
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM419
h-miR-371
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM420
h-miR-372
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM421
h-miR-373
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM422
h-miR-373*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM423
h-miR-374
6.00
6.69
6.19
7.70
7.86
6.00
6.69
6.60
7.31



EAM426
m-miR-215
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM427
hm-miR-409-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM428
hm-miR-410
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM429
m-miR-376b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM430
m-miR-376a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM431
m-miR-411
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM432
m-miR-380-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM433
hm-miR-412
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM264
hmr-miR-27b
7.18
6.00
6.78
7.56
8.20
6.00
6.00
6.80
7.55



EAM263
hmr-miR-26a
9.87
9.50
10.10
9.97
9.68
9.23
9.40
10.04
10.07



EAM262
hmr-miR-24
6.00
6.25
6.00
6.94
6.00
6.00
6.00
6.00
7.29



EAM261
hmr-miR-23b
7.42
7.66
7.78
8.90
8.11
7.19
9.17
8.50
7.90



EAM260
hmr-miR-23a
7.78
8.15
7.85
9.30
7.94
8.13
8.87
8.83
8.09



EAM256
h-miR-220
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM255
hmr-miR-22
6.00
6.24
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM248
hmr-miR-213
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM244
hmr-miR-21
6.39
7.99
8.45
8.89
8.32
9.27
8.02
7.77
7.86



EAM240
hmr-miR-20a
11.36
11.38
11.87
11.08
11.27
11.19
11.27
11.27
11.50



EAM237
hmr-miR-19b
8.88
7.38
9.64
9.13
8.68
8.34
8.68
8.09
9.38



EAM233
hmr-miR-196a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM214
hm-miR-148a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM212
hmr-miR-145
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM211
hmr-miR-144
9.13
7.29
6.00
6.37
6.88
7.86
6.00
6.00
6.00



EAM210
hmr-miR-143
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM389
r-miR-346
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM390
r-miR-347
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM391
r-miR-349
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA223
hmr-miR-33
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM277
hmr-miR-96
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM276
hmr-miR-9
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM272
hmr-miR-30d
7.05
6.00
7.40
8.82
8.29
8.37
8.23
8.50
8.82



EAM288
mr-miR-10b
6.74
8.16
6.94
6.48
7.41
6.00
6.56
6.00
6.00



EAM293
hm-miR-188
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM297
hmr-miR-193a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM301
h-miR-198
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM232
hmr-miR-192
6.11
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM231
hmr-miR-187
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM230
hmr-miR-183
6.00
6.00
6.00
6.00
6.29
6.00
6.00
6.00
6.00



EAM229
hm-miR-182
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM220
hmr-miR-154
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM219
hmr-miR-153
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM218
hmr-miR-152
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM217
hmr-miR-150
6.00
7.22
6.00
6.00
6.00
7.52
6.00
6.00
6.78



EAM216
hm-miR-149
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM215
hmr-miR-148b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM271
hmr-miR-30c
7.66
7.76
8.70
9.30
9.01
9.25
9.17
9.58
8.66



EAM268
hmr-miR-29a
6.19
7.88
6.00
6.00
6.00
6.68
6.00
6.00
6.90



EAM305
hmr-miR-200b
6.00
6.00
6.00
6.00
6.28
6.00
7.02
6.00
6.46



EAM303
hm-miR-199a*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM300
h-miR-197
7.59
6.00
6.82
7.62
8.19
7.13
6.99
8.12
8.11



EAM299
hmr-miR-195
9.34
9.94
9.20
8.70
8.93
9.57
8.49
8.73
9.15



JLA91
hmr-miR-99b
9.43
8.63
6.00
9.99
7.89
8.23
7.06
7.31
6.00



JLA92
hmr-miR-433
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA93
hmr-miR-431
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA94
hmr-miR-365
8.38
6.00
8.96
7.30
7.15
6.00
6.43
6.00
6.00



JLA95
hmr-miR-450
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA96
hmr-miR-449
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA99
hmr-miR-448
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA103
hmr-miR-424
6.00
6.20
6.81
7.28
6.00
6.00
6.66
6.00
7.64



JLA105
hm-miR-361
6.00
6.01
6.00
7.74
7.07
7.28
7.99
6.00
7.46



JLA106
hm-miR-375
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA107
hm-miR-377
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA108
hm-miR-378
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA109
hm-miR-379
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA110
hm-miR-380-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA111
hm-miR-381
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA112
hm-miR-382
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA115
hm-miR-384
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA116
hm-miR-425
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA117
hm-miR-452
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA118
hm-miR-30e-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA104
mr-miR-129-3p
9.27
6.00
9.25
6.00
8.52
6.00
7.63
7.12
6.00



JLA98
mr-miR-429
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA101
mr-miR-330
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA102
mr-miR-322
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA114
m-miR-383
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA5
hmr-miR-451
9.95
8.32
6.00
6.85
9.54
8.59
6.00
6.33
6.00



JLA201
r-miR-421
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA202
m-miR-463
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA203
m-miR-464
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA204
m-miR-465
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA205
m-miR-466
7.94
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA206
m-miR-467
8.36
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA207
m-miR-468
6.05
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA208
m-miR-469
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA209
m-miR-470
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA210
m-miR-471
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM325
hmr-miR-27a
8.16
6.00
7.67
8.75
8.56
6.00
6.00
7.93
8.44



EAM326
hmr-miR-296
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM327
hmr-miR-299-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM328
hmr-miR-301
6.25
6.00
6.00
6.00
6.00
6.00
6.00
6.56
6.16



EAM329
hm-miR-302a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM330
hmr-miR-30a-5p
6.00
6.01
6.00
7.29
6.25
6.55
6.00
6.78
7.23



EAM331
hmr-miR-30e-5p
6.00
6.21
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM332
hmr-miR-31
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM333
hmr-miR-32
7.38
6.00
6.21
6.00
6.00
6.00
6.00
6.00
6.00



EAM335
h-miR-34b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM336
hmr-miR-34c
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM337
hmr-miR-93
8.93
9.74
9.14
9.92
8.36
10.06
10.35
9.58
10.28



EAM208
hmr-miR-141
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM207
hmr-miR-140
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA222
hmr-miR-139
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA220
hmr-miR-138
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM203
hmr-miR-135a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM200
hmr-miR-133a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM
hmr-miR-128b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM
hmr-miR-128a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM254
hmr-miR-219
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM257
hmr-miR-221
7.93
6.55
6.23
6.00
6.00
6.00
6.00
6.00
6.29



EAM258
hmr-miR-222
6.00
8.99
9.28
8.07
8.88
8.98
9.45
8.55
8.72



EAM259
hmr-miR-223
7.82
7.94
7.94
8.62
7.50
8.85
8.28
8.36
8.42



JLA211
m-miR-434-5p
6.65
6.49
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA212
m-miR-434-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA213
m-miR-433-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA2
hsa-miR-522
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA3
hsa-miR-495
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA200
r-miR-297
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA6
hsa-miR-518e
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA7
hsa-miR-519a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA8
hsa-mir-527*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA72
hmr-miR-140*
8.13
6.00
6.00
7.81
7.88
6.14
6.00
6.44
6.00



JLA10
hsa-miR-521
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA12
hsa-miR-362
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA74
hsa-mir-18*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA14
hm-miR-363
6.31
6.77
6.00
7.17
6.26
6.00
6.00
7.78
6.35



JLA77
hsa-mir-19b-1*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA17
hsa-mir-
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00




520c,b,f












JLA79
hsa-mir-23a*
6.00
7.27
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA20
hsa-miR-369-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA81
hsa-mir-339*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA23
hsa-mir-342*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.10
6.00



JLA24
hsa-mir-19a*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA26
hsa-miR-517a,b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
7.45



JLA27
hsa-miR-516-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.26



JLA28
hsa-miR-518b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA29
hsa-miR-519d
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.43



JLA73
hr-mir-151*
7.12
6.00
6.00
6.00
7.45
7.00
7.48
6.00
6.35



JLA31
hsa-mir-28*
6.00
6.00
6.00
6.31
6.00
6.00
6.00
6.00
6.00



JLA33
hsa-mir-519a-2*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA34
hsa-mir-26b*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA35
hsa-miR-526c
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA36
hsa-miR-527
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA38
hsa-mir-29b-2*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA39
hsa-let-7g*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA40
hsa-miR-518a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA41
hsa-miR-523
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA44
hsa-miR-515-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA45
hsa-mir-146b*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA49
hsa-mir-222*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA53
hsa-mir-24*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA55
hsa-miR-503
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA57
hsa-mir-505
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA82
hsa-mir-423*
10.00
10.80
10.94
10.76
10.93
10.75
10.65
10.87
10.46



JLA66
hsa-miR-432
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA83
hsa-mir-425*
6.00
6.00
6.77
7.81
8.68
7.65
7.36
6.86
6.00



JLA84
hsa-mir-92-1*
6.00
6.00
6.00
7.03
6.00
6.00
6.00
6.00
6.00



JLA69
hsa-mir-193*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA70
hsa-miR-515-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA71
hsa-mir-516-1*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA85
hsa-mir-30d*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA125
h-miR-20b
10.18
10.08
10.29
9.65
9.78
10.07
9.95
10.22
10.08



JLA198
h-miR-191*
6.00
6.00
6.00
6.00
6.00
6.36
6.00
6.00
6.00



JLA199
h-miR-154*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM316
h-miR-147
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM317
h-miR-155
9.59
10.47
9.67
10.45
9.61
9.58
9.48
9.42
9.75



EAM318
h-miR-17-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
7.19
6.00



JLA195
h-miR-200a*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA196
h-miR-302a*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA197
h-miR-299-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
7.25



EAM319
h-miR-182*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM405
h-miR-302b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM406
h-miR-302b*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



EAM392
r-miR-352
6.25
7.91
7.30
8.16
7.22
8.24
7.61
7.55
7.24



JLA123
h-miR-423
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



JLA124
h-miR-18b
6.00
6.00
6.00
7.59
6.00
6.00
6.00
8.06
8.14




















ProbeID
Description
ERY1_2
ERY1_3
ERY1_4
ERY2_1
ERY2_2
ERY2_3
ERY3_1
ERY3_2
MEGA1_1





EAM190
h-miR-10b
6.00
6.99
6.75
6.00
6.00
7.96
6.06
6.00
6.00


EAM187
hmr-miR-107
8.25
6.00
7.84
6.00
7.81
6.90
6.00
7.49
8.75


EAM185
hmr-miR-103
8.79
6.00
8.39
6.00
8.34
7.93
6.00
7.79
9.02


EAM181
hmr-let-7f
7.95
8.58
8.86
9.12
9.72
6.00
9.09
8.47
9.80


EAM179
hmr-let-7d
7.02
7.49
8.73
7.65
9.88
8.43
9.02
9.01
9.57


EAM177
mr-miR-101b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM175
hmr-miR-320
9.89
9.23
8.71
8.40
10.08
7.51
6.66
7.03
8.58


EAM168
hmr-let-7e
6.00
6.93
6.00
6.00
6.62
6.00
6.00
6.00
6.00


EAM161
hmr-miR-28
6.00
6.00
6.00
6.00
6.08
6.00
6.00
6.00
7.52


EAM160
hmr-miR-26b
8.10
8.96
9.69
9.37
9.42
7.41
8.32
7.48
9.69


EAM155
hmr-miR-136
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM283
mr-miR-211
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM282
m-miR-199b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM281
mr-miR-217
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM280
hmr-miR-30a-
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00



3p











EAM279
hmr-miR-29c
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM278
hmr-miR-98
6.00
6.00
6.00
6.25
6.00
6.00
6.00
6.00
6.00


EAM270
hmr-miR-30b
8.99
7.63
8.95
8.53
9.45
7.95
7.36
6.00
9.58


EAM159
hmr-miR-130a
8.43
8.44
7.06
6.00
8.49
9.20
6.00
6.00
9.15


EAM163
hmr-miR-142-
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
7.47



3p











EAM171
hmr-miR-137
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM306
m-miR-201
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM307
m-miR-202
6.00
6.00
6.00
6.00
6.00
7.33
6.67
6.00
6.00


EAM308
hmr-miR-206
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM309
m-miR-207
6.00
6.00
6.00
7.37
6.00
6.00
6.00
6.00
6.00


EAM310
hmr-miR-208
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM247
hmr-miR-212
6.00
6.37
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM251
hmr-miR-216
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM253
hmr-miR-218
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM275
hmr-miR-34a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM246
h-miR-211
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM250
h-miR-215
6.00
6.00
6.92
6.00
6.00
6.00
6.40
7.44
6.00


EAM252
h-miR-217
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM224
hmr-miR-17-5p
11.52
10.93
10.34
10.89
10.35
11.50
10.47
10.31
10.24


EAM225
hmr-miR-18a
7.13
7.50
6.00
6.00
6.00
7.18
6.00
6.00
6.00


EAM226
hmr-miR-181a
7.91
9.01
7.64
6.00
8.20
6.00
6.00
6.00
8.26


EAM227
hmr-miR-181b
6.95
7.06
6.00
6.00
7.97
6.00
6.00
6.00
6.00


EAM234
hmr-miR-199a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM235
h-miR-199b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM236
hmr-miR-19a
7.76
8.02
7.53
8.50
7.41
7.49
6.00
7.84
7.23


EAM241
hmr-miR-203
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM242
hmr-miR-204
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM243
hmr-miR-205
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.69


EAM245
hmr-miR-210
6.00
6.00
6.48
6.00
6.00
6.00
6.00
6.00
6.00


EAM249
hmr-miR-214
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM184
hmr-miR-100
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM186
h-miR-106a
11.18
10.83
10.11
10.62
10.16
11.33
10.18
10.13
10.04


EAM189
hmr-miR-10a
6.00
7.49
9.10
6.00
6.43
6.00
6.00
6.00
6.54


EAM191
hmr-miR-122a
6.00
6.00
7.20
6.00
6.00
6.00
6.00
6.00
6.00


EAM192
hmr-miR-126*
7.58
8.43
8.67
6.00
6.00
6.00
6.00
6.00
8.11


EAM198
hmr-miR-130b
6.00
6.01
6.00
6.00
7.44
6.00
6.00
7.98
6.00


EAM202
hmr-miR-134
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM209
hmr-miR-142-5p
8.13
6.00
6.75
6.77
7.48
8.09
6.00
6.00
9.02


EAM221
m-miR-155
7.36
7.54
6.00
6.59
7.89
6.00
6.00
6.00
6.00


EAM223
hmr-miR-15b
10.30
9.57
10.03
11.29
10.88
11.16
11.83
11.90
11.05


EAM228
hmr-miR-181c
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM222
hm-miR-15a
7.27
8.26
6.00
8.36
9.42
10.08
10.72
9.83
8.88


EAM111
hm-let-7g
8.71
9.39
9.63
9.43
10.51
10.13
6.00
9.81
10.39


EAM131
hmr-miR-92
11.87
11.49
9.87
11.68
11.05
9.45
11.53
11.40
8.71


EAM139
hmr-miR-146a
7.16
9.30
8.85
6.00
9.34
10.46
6.82
6.00
10.20


EAM145
hmr-let-7c
8.87
10.13
10.11
9.49
9.50
9.31
8.41
8.87
8.35


EAM109
hmr-miR-7
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM152
hm-miR-9*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA215
hmr-let-7i
6.00
6.35
9.31
7.17
8.40
9.25
6.00
7.09
8.55


EAM153
hmr-let-7a
10.73
11.16
12.01
10.47
10.87
11.26
10.78
11.14
10.70


EAM147
hmr-let-7b
7.57
9.14
8.77
9.57
9.66
6.51
7.43
7.57
8.10


EAM137
hmr-miR-132
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM133
hmr-miR-324-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM103
hmr-miR-124a
6.00
6.00
6.00
6.00
6.00
6.21
6.00
6.00
6.00


EAM105
hmr-miR-125b
7.21
7.24
7.48
6.00
6.34
6.00
6.00
6.23
6.00


EAM121
hmr-miR-99a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM115
hmr-miR-16
12.08
12.26
12.61
12.97
12.35
12.37
13.12
12.85
12.67


EAM119
hmr-miR-29b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM311
hmr-miR-101
6.00
6.24
6.18
6.00
6.00
6.00
6.00
6.00
6.00


EAM312
h-miR-105
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM313
hmr-miR-106b
8.64
8.72
9.09
9.65
7.84
9.00
8.32
8.32
8.32


EAM314
hmr-miR-126
10.18
11.18
11.85
7.84
9.11
6.00
6.00
6.00
7.96


EAM315
hmr-miR-127
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM320
hm-miR-189
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA216
hmr-miR-200c
6.00
6.00
6.00
6.00
7.88
6.00
6.00
6.00
6.00


EAM323
h-miR-224
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.49


EAM324
hmr-miR-25
8.01
8.81
6.04
7.43
8.29
6.00
6.00
8.55
7.79


EAM386
r-miR-336
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA218
r-miR-343
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM388
r-miR-344
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM338
h-miR-95
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA214
hmr-miR-129
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM340
mr-let-7d*
6.00
7.18
6.00
6.00
6.00
6.00
6.00
8.01
6.00


EAM341
m-miR-106a
10.77
10.26
10.07
10.38
9.39
10.73
9.85
9.85
9.54


EAM342
hmr-miR-135b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM343
mr-miR-151
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM344
m-miR-17-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM345
m-miR-224
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM346
mr-miR-290
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM347
mr-miR-291-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM348
mr-miR-291-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM349
mr-miR-292-3p
6.00
6.00
7.71
6.00
6.00
6.00
6.00
6.00
6.00


EAM350
mr-miR-292-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM351
m-miR-293
6.00
6.00
6.00
8.36
6.00
6.45
6.69
6.00
6.00


EAM352
m-miR-294
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM353
m-miR-295
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM354
m-miR-297
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM355
mr-miR-298
6.00
6.00
6.00
6.00
6.00
6.00
10.24
6.00
6.00


EAM356
mr-miR-300
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM358
hmr-miR-323
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM359
hmr-miR-324-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM360
mr-miR-325
6.00
6.00
7.44
6.00
6.00
6.39
6.00
6.00
6.00


EAM361
hmr-miR-326
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM362
hmr-miR-328
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM363
mr-miR-329
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM365
hmr-miR-331
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM366
mr-miR-337
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM367
hmr-miR-338
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM368
hmr-miR-339
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM369
hmr-miR-340
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM370
mr-miR-341
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM371
hmr-miR-342
7.83
6.00
6.00
9.42
10.64
9.88
6.00
9.57
10.38


EAM372
m-miR-344
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM373
mr-miR-345
6.00
6.00
6.00
6.00
6.00
6.00
7.09
6.00
6.00


EAM374
m-miR-346
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM375
mr-miR-34b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA217
mr-miR-350
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM377
mr-miR-351
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM378
mr-miR-7b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM382
r-miR-20*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM383
r-miR-327
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM384
r-miR-333
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM385
hmr-miR-335
7.14
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM393
r-miR-7*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM304
hmr-miR-200a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM298
hmr-miR-194
6.07
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.48


JLA221
hmr-miR-191
8.31
6.00
6.60
8.62
7.95
7.15
8.30
8.00
8.50


EAM295
hmr-miR-190
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM292
hmr-miR-186
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA219
hmr-miR-185
6.00
7.21
6.00
6.54
7.12
6.00
7.47
9.59
6.23


EAM290
hmr-miR-184
6.00
6.21
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM402
hm-miR-133b
6.00
6.00
7.29
6.00
6.00
6.00
6.00
6.00
6.00


EAM403
h-miR-151
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM404
hmr-miR-196b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM418
hm-miR-370
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM419
h-miR-371
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM420
h-miR-372
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM421
h-miR-373
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM422
h-miR-373*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM423
h-miR-374
6.00
7.19
7.18
6.25
7.10
6.61
6.00
6.00
6.70


EAM426
m-miR-215
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM427
hm-miR-409-3p
6.54
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM428
hm-miR-410
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM429
m-miR-376b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM430
m-miR-376a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM431
m-miR-411
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM432
m-miR-380-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM433
hm-miR-412
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM264
hmr-miR-27b
6.00
6.00
8.78
6.80
6.95
8.02
6.00
7.45
8.57


EAM263
hmr-miR-26a
9.27
9.43
9.90
9.27
9.26
8.54
8.77
8.20
10.49


EAM262
hmr-miR-24
6.00
6.00
8.39
7.36
6.00
6.00
6.00
6.00
6.00


EAM261
hmr-miR-23b
7.59
8.64
8.06
7.72
8.59
8.34
6.92
7.59
8.07


EAM260
hmr-miR-23a
8.42
9.19
8.03
8.11
8.60
8.55
6.00
6.00
8.15


EAM256
h-miR-220
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM255
hmr-miR-22
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.46


EAM248
hmr-miR-213
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM244
hmr-miR-21
8.34
8.60
6.73
6.00
8.29
7.93
6.00
6.00
9.48


EAM240
hmr-miR-20a
11.40
11.35
9.92
11.02
10.86
12.04
10.43
10.13
10.28


EAM237
hmr-miR-19b
8.65
9.13
8.54
9.35
8.39
8.54
6.00
8.44
7.57


EAM233
hmr-miR-196a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM214
hm-miR-148a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM212
hmr-miR-145
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.40


EAM211
hmr-miR-144
6.00
6.00
6.00
8.80
6.00
8.40
11.13
10.47
7.05


EAM210
hmr-miR-143
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM389
r-miR-346
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM390
r-miR-347
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM391
r-miR-349
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA223
hmr-miR-33
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM277
hmr-miR-96
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM276
hmr-miR-9
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM272
hmr-miR-30d
8.59
6.70
8.78
7.19
8.38
6.00
6.00
6.04
8.35


EAM288
mr-miR-10b
6.00
6.00
7.04
6.00
6.00
7.72
6.00
6.98
6.00


EAM293
hm-miR-188
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM297
hmr-miR-193a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM301
h-miR-198
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM232
hmr-miR-192
6.00
6.00
6.00
6.00
6.00
6.00
7.14
7.65
6.00


EAM231
hmr-miR-187
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM230
hmr-miR-183
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM229
hm-miR-182
6.00
6.00
6.00
6.39
6.00
6.00
6.00
6.00
6.00


EAM220
hmr-miR-154
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM219
hmr-miR-153
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM218
hmr-miR-152
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.74


EAM217
hmr-miR-150
6.00
6.00
8.16
6.79
9.98
6.00
6.00
6.15
10.77


EAM216
hm-miR-149
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM215
hmr-miR-148b
6.00
6.83
6.00
6.00
6.00
6.00
6.00
6.00
7.49


EAM271
hmr-miR-30c
9.13
7.46
8.56
8.72
9.62
7.65
7.67
6.00
9.52


EAM268
hmr-miR-29a
6.00
6.00
6.85
6.00
6.20
6.00
6.00
6.00
6.00


EAM305
hmr-miR-200b
6.00
6.00
6.00
6.00
7.01
6.00
6.00
6.00
6.00


EAM303
hm-miR-199a*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM300
h-miR-197
8.29
8.88
7.83
8.40
8.70
6.00
6.00
6.84
7.76


EAM299
hmr-miR-195
9.77
9.49
9.17
10.48
9.03
9.81
10.31
9.58
9.72


JLA91
hmr-miR-99b
10.42
6.63
8.77
6.00
6.00
6.00
6.00
6.00
6.00


JLA92
hmr-miR-433
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA93
hmr-miR-431
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA94
hmr-miR-365
6.06
7.03
7.03
6.00
6.00
8.39
6.22
6.53
8.13


JLA95
hmr-miR-450
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA96
hmr-miR-449
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA99
hmr-miR-448
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA103
hmr-miR-424
6.00
9.39
9.21
6.98
6.00
6.00
6.00
6.00
6.00


JLA105
hm-miR-361
6.00
8.25
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA106
hm-miR-375
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA107
hm-miR-377
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA108
hm-miR-378
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA109
hm-miR-379
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA110
hm-miR-380-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA111
hm-miR-381
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA112
hm-miR-382
8.83
6.00
6.00
6.00
7.08
6.00
6.00
6.00
6.00


JLA115
hm-miR-384
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA116
hm-miR-425
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA117
hm-miR-452
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA118
hm-miR-30e-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA104
mr-miR-129-3p
7.18
6.00
6.00
6.24
6.00
7.62
8.58
7.16
7.12


JLA98
mr-miR-429
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA101
mr-miR-330
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA102
mr-miR-322
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA114
m-miR-383
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA5
hmr-miR-451
6.00
6.15
7.06
9.77
7.29
6.77
11.41
12.04
6.79


JLA201
r-miR-421
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA202
m-miR-463
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA203
m-miR-464
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA204
m-miR-465
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA205
m-miR-466
6.00
6.00
6.00
6.00
6.00
7.54
6.48
6.00
6.00


JLA206
m-miR-467
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA207
m-miR-468
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA208
m-miR-469
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA209
m-miR-470
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA210
m-miR-471
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM325
hmr-miR-27a
6.00
6.00
8.84
7.51
7.75
9.09
6.00
6.80
9.54


EAM326
hmr-miR-296
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM327
hmr-miR-299-
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM328
hmr-miR-301
6.00
6.00
6.00
6.09
6.00
6.00
6.00
6.00
6.00


EAM329
hm-miR-302a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM330
hmr-miR-30a-
6.05
6.84
8.16
6.64
6.71
6.00
6.00
6.00
6.62


EAM331
hmr-miR-30e-
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM332
hmr-miR-31
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM333
hmr-miR-32
6.00
7.53
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM335
h-miR-34b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM336
hmr-miR-34c
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM337
hmr-miR-93
9.86
7.32
6.00
9.38
9.47
8.20
7.82
10.69
8.89


EAM208
hmr-miR-141
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM207
hmr-miR-140
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA222
hmr-miR-139
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA220
hmr-miR-138
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM203
hmr-miR-135a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM200
hmr-miR-133a
6.00
6.00
7.18
6.00
6.00
6.00
6.00
6.00
6.00


EAM
hmr-miR-128b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM
hmr-miR-128a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM254
hmr-miR-219
6.00
6.00
6.00
6.00
6.00
6.00
6.45
6.00
6.00


EAM257
hmr-miR-221
6.00
7.88
6.00
6.00
6.00
6.00
8.25
6.00
8.79


EAM258
hmr-miR-222
6.90
7.15
7.46
7.68
8.11
6.00
6.00
6.00
8.38


EAM259
hmr-miR-223
8.26
6.46
6.31
9.11
8.85
8.89
6.00
6.00
8.82


JLA211
m-miR-434-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA212
m-miR-434-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA213
m-miR-433-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA2
hsa-miR-522
6.00
6.00
6.86
6.00
6.00
6.00
6.00
6.00
6.00


JLA3
hsa-miR-495
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA200
r-miR-297
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA6
hsa-miR-518e
6.00
6.00
6.00
6.00
6.00
6.00
6.58
6.67
6.00


JLA7
hsa-miR-519a
6.00
6.80
7.83
6.00
6.00
6.00
6.68
6.53
6.00


JLA8
hsa-mir-527*
6.00
6.00
6.00
6.00
6.00
6.00
6.19
6.07
6.00


JLA72
hmr-miR-140*
6.00
6.00
6.01
6.00
8.10
6.00
6.71
7.22
7.30


JLA10
hsa-miR-521
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA12
hsa-miR-362
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA74
hsa-mir-18*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA14
hm-miR-363
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA77
hsa-mir-19b-1*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA17
hsa-mir-
6.00
6.00
6.69
6.00
6.00
6.00
6.00
6.00
6.00



520c,b,f











JLA79
hsa-mir-23a*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA20
hsa-miR-369-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA81
hsa-mir-339*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA23
hsa-mir-342*
7.31
6.00
6.00
6.00
6.00
6.00
6.00
6.00
8.07


JLA24
hsa-mir-19a*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA26
hsa-miR-517a,b
9.19
9.68
9.88
6.00
6.00
6.00
6.00
6.00
6.00


JLA27
hsa-miR-516-5p
8.01
7.22
8.30
6.00
6.00
6.00
6.00
6.00
6.00


JLA28
hsa-miR-518b
6.00
6.00
6.10
6.00
6.00
6.00
6.00
6.00
6.00


JLA29
hsa-miR-519d
6.00
8.31
9.35
6.00
6.00
6.00
6.00
6.00
6.00


JLA73
hr-mir-151*
8.47
6.00
6.00
6.00
7.04
6.00
6.03
8.07
7.32


JLA31
hsa-mir-28*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA33
hsa-mir-519a-2*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA34
hsa-mir-26b*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA35
hsa-miR-526c
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA36
hsa-miR-527
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA38
hsa-mir-29b-2*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA39
hsa-let-7g*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA40
hsa-miR-518a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA41
hsa-miR-523
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA44
hsa-miR-515-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA45
hsa-mir-146b*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA49
hsa-mir-222*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA53
hsa-mir-24*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA55
hsa-miR-503
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA57
hsa-mir-505
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA82
hsa-mir-423*
10.59
11.08
10.84
10.71
10.99
9.16
12.28
11.26
10.01


JLA66
hsa-miR-432
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA83
hsa-mir-425*
6.69
9.11
6.64
8.35
7.69
6.00
6.09
6.00
8.19


JLA84
hsa-mir-92-1*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA69
hsa-mir-193*
6.00
6.00
6.00
6.00
6.00
6.88
7.72
6.00
6.00


JLA70
hsa-miR-515-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA71
hsa-mir-516-1*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA85
hsa-mir-30d*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA125
h-miR-20b
10.33
10.27
8.32
9.68
9.66
11.07
9.22
8.63
8.99


JLA198
h-miR-191*
6.30
6.00
6.00
7.24
6.00
6.58
6.63
6.11
6.26


JLA199
h-miR-154*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM316
h-miR-147
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM317
h-miR-155
8.97
9.50
7.69
8.48
9.68
7.96
6.18
6.00
6.00


EAM318
h-miR-17-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA195
h-miR-200a*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA196
h-miR-302a*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA197
h-miR-299-3p
6.41
6.00
6.82
6.00
6.78
7.18
6.47
6.00
6.00


EAM319
h-miR-182*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM405
h-miR-302b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM406
h-miR-302b*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM392
r-miR-352
6.00
6.00
6.96
6.00
8.26
7.14
6.72
7.48
7.86


JLA123
h-miR-423
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.39
6.00


JLA124
h-miR-18b
7.00
7.20
6.00
6.00
6.00
7.50
6.00
6.00
6.00






















MEGA1—
MEGA1—
MEGA1—
MEGA2—







ProbeID
Description
2
3
4
1
MEGA2_2
MEGA2_3
MEGA2_4
MEGA2_5
MEGA2_6





EAM190
h-miR-10b
6.00
6.00
6.00
6.00
6.00
6.66
6.00
6.00
7.22


EAM187
hmr-miR-107
6.30
6.68
6.79
6.00
6.00
8.32
7.63
6.92
8.79


EAM185
hmr-miR-103
6.76
7.19
7.67
6.44
6.00
8.54
8.04
7.13
9.39


EAM181
hmr-let-7f
9.53
8.57
10.11
6.73
8.73
9.75
8.60
9.07
9.57


EAM179
hmr-let-7d
9.31
8.27
8.89
9.64
9.34
9.91
9.61
9.83
9.79


EAM177
mr-miR-101b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM175
hmr-miR-320
8.34
8.72
8.73
8.11
8.87
8.50
8.70
7.50
7.51


EAM168
hmr-let-7e
6.00
6.00
8.34
6.65
6.00
6.00
6.00
6.00
7.50


EAM161
hmr-miR-28
7.96
6.00
8.29
8.44
8.09
8.01
7.47
6.00
8.47


EAM160
hmr-miR-26b
9.65
8.59
9.78
8.86
9.24
9.38
9.23
8.61
8.95


EAM155
hmr-miR-136
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM283
mr-miR-211
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM282
m-miR-199b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM281
mr-miR-217
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM280
hmr-miR-30a-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM279
hmr-miR-29c
6.56
6.00
6.00
6.00
6.16
8.18
7.23
6.00
6.00


EAM278
hmr-miR-98
8.00
7.69
7.32
8.11
6.00
6.00
7.17
9.06
6.50


EAM270
hmr-miR-30b
8.74
8.82
9.46
8.10
8.74
9.66
9.54
6.00
9.43


EAM159
hmr-miR-130a
7.39
8.91
6.79
8.20
7.67
7.48
6.87
6.00
6.00


EAM163
hmr-miR-142-3p
7.43
6.00
7.89
7.56
6.00
7.79
7.27
8.56
6.00


EAM171
hmr-miR-137
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM306
m-miR-201
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM307
m-miR-202
6.00
6.00
6.00
7.21
6.00
6.00
6.00
7.05
6.00


EAM308
hmr-miR-206
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM309
m-miR-207
6.00
6.00
7.57
6.00
6.00
6.00
6.00
6.00
6.00


EAM310
hmr-miR-208
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM247
hmr-miR-212
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM251
hmr-miR-216
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM253
hmr-miR-218
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM275
hmr-miR-34a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM246
h-miR-211
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM250
h-miR-215
6.00
6.00
6.00
6.00
6.00
6.00
6.00
7.32
6.00


EAM252
h-miR-217
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM224
hmr-miR-17-5p
10.44
11.27
9.91
10.39
8.79
10.13
10.33
9.48
9.88


EAM225
hmr-miR-18a
7.40
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM226
hmr-miR-181a
7.87
8.68
7.08
6.00
8.81
6.84
9.72
6.00
6.56


EAM227
hmr-miR-181b
6.79
7.49
6.00
6.00
6.00
6.00
7.54
6.00
6.00


EAM234
hmr-miR-199a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM235
h-miR-199b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM236
hmr-miR-19a
7.70
8.90
8.55
6.00
7.14
6.00
7.61
8.19
6.60


EAM241
hmr-miR-203
6.00
8.15
6.99
6.00
6.00
6.00
6.00
8.16
6.00


EAM242
hmr-miR-204
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM243
hmr-miR-205
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM245
hmr-miR-210
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM249
hmr-miR-214
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.12


EAM184
hmr-miR-100
6.00
6.00
6.00
6.00
6.00
6.00
6.00
7.20
6.00


EAM186
h-miR-106a
10.31
10.92
9.89
10.11
8.23
10.12
10.01
9.48
9.71


EAM189
hmr-miR-10a
7.74
8.59
8.29
6.00
6.00
6.00
6.72
6.00
6.00


EAM191
hmr-miR-122a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM192
hmr-miR-126*
6.82
7.25
6.18
6.97
6.00
6.00
6.00
6.00
7.21


EAM198
hmr-miR-130b
6.27
6.21
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM202
hmr-miR-134
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM209
hmr-miR-142-5p
7.81
7.04
7.93
7.90
6.86
8.66
8.37
9.90
7.87


EAM221
m-miR-155
7.17
8.01
7.16
6.00
6.00
7.48
6.00
6.00
7.03


EAM223
hmr-miR-15b
10.50
10.40
9.74
11.50
11.22
10.39
10.79
11.03
11.04


EAM228
hmr-miR-181c
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM222
hm-miR-15a
9.52
9.67
6.00
9.33
6.00
8.87
10.18
10.57
8.69


EAM111
hm-let-7g
10.55
10.12
10.44
8.32
9.39
11.19
10.14
10.65
10.03


EAM131
hmr-miR-92
10.50
11.33
9.41
9.74
11.07
9.10
10.74
9.01
10.31


EAM139
hmr-miR-146a
9.47
10.48
8.86
6.00
8.51
9.66
8.61
6.00
8.90


EAM145
hmr-let-7c
8.69
9.07
9.10
7.53
9.60
7.46
6.68
9.21
7.82


EAM109
hmr-miR-7
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM152
hm-miR-9*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA215
hmr-let-7i
8.71
8.89
9.07
9.12
8.59
8.08
9.20
10.60
9.03


EAM153
hmr-let-7a
10.98
10.75
11.12
10.65
11.41
10.93
10.44
11.51
11.17


EAM147
hmr-let-7b
8.34
7.17
10.57
6.00
6.58
6.09
6.78
9.59
6.11


EAM 137
hmr-miR-132
6.00
6.95
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM133
hmr-miR-324-5p
6.00
6.00
6.00
6.00
6.00
6.00
7.17
6.00
6.00


EAM103
hmr-miR-124a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM105
hmr-miR-125b
6.00
7.61
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM121
hmr-miR-99a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
7.94
6.00


EAM115
hmr-miR-16
12.56
12.12
13.06
12.43
12.37
12.79
12.11
13.02
12.69


EAM119
hmr-miR-29b
6.00
6.00
6.91
6.00
6.00
6.00
7.01
6.00
6.00


EAM311
hmr-miR-101
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM312
h-miR-105
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM313
hmr-miR-106b
8.85
9.53
9.17
8.97
8.11
9.38
8.94
7.61
8.82


EAM314
hmr-miR-126
9.23
10.02
9.48
8.22
6.00
8.38
8.42
6.00
8.73


EAM315
hmr-miR-127
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM320
hm-miR-189
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA216
hmr-miR-200c
6.00
6.00
6.00
6.96
6.00
7.14
6.52
7.22
6.00


EAM323
h-miR-224
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM324
hmr-miR-25
6.85
8.56
6.00
6.00
7.38
6.00
7.51
6.00
6.00


EAM386
r-miR-336
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA218
r-miR-343
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM388
r-miR-344
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM338
h-miR-95
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA214
hmr-miR-129
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM340
mr-let-7d*
6.00
6.00
6.00
6.99
6.00
6.00
6.00
6.00
6.00


EAM341
m-miR-106a
9.43
10.30
9.16
9.99
8.43
9.69
9.84
7.93
9.04


EAM342
hmr-miR-135b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM343
mr-miR-151
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM344
m-miR-17-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM345
m-miR-224
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM346
mr-miR-290
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM347
mr-miR-291-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM348
mr-miR-291-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM349
mr-miR-292-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM350
mr-miR-292-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM351
m-miR-293
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM352
m-miR-294
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM353
m-miR-295
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM354
m-miR-297
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM355
mr-miR-298
6.00
6.00
6.00
6.00
6.00
7.52
6.00
6.00
6.00


EAM356
mr-miR-300
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM358
hmr-miR-323
6.00
6.00
6.49
6.00
6.00
6.00
6.00
6.00
6.00


EAM359
hmr-miR-324-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM360
mr-miR-325
6.00
6.63
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM361
hmr-miR-326
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM362
hmr-miR-328
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM363
mr-miR-329
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM365
hmr-miR-331
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM366
mr-miR-337
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM367
hmr-miR-338
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM368
hmr-miR-339
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM369
hmr-miR-340
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM370
mr-miR-341
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM371
hmr-miR-342
11.21
8.81
10.24
11.01
11.98
10.79
11.69
11.33
11.20


EAM372
m-miR-344
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM373
mr-miR-345
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM374
m-miR-346
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM37
mr-miR-34b
6.00
6.00
6.00
6.19
6.00
6.00
6.00
6.00
6.00


JLA217
mr-miR-350
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM37
mr-miR-351
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM37
mr-miR-7b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM38
r-miR-20*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM38
r-miR-327
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM38
r-miR-333
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM38
hmr-miR-335
7.18
7.80
6.00
6.00
6.00
6.42
6.00
6.00
6.00


EAM39
r-miR-7*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM30
hmr-miR-200a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM29
hmr-miR-194
6.00
6.22
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA221
hmr-miR-191
8.20
7.45
8.85
7.58
8.83
7.67
8.32
8.13
8.89


EAM29
hmr-miR-190
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM29
hmr-miR-186
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA219
hmr-miR-185
6.00
6.00
6.00
6.00
6.00
6.00
7.40
6.00
6.00


EAM29
hmr-miR-184
6.00
6.00
6.00
6.00
6.00
6.00
7.33
6.00
6.00


EAM40
hm-miR-133b
6.00
6.00
6.00
6.94
6.00
6.00
6.00
6.00
6.00


EAM40
h-miR-151
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM40
hmr-miR-196b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM41
hm-miR-370
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM41
h-miR-371
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM42
h-miR-372
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM42
h-miR-373
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM42
h-miR-373*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM42
h-miR-374
6.00
7.84
6.58
6.50
6.70
6.78
7.50
6.00
6.82


EAM42
m-miR-215
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM42
hm-miR-409-3p
6.80
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM42
hm-miR-410
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM42
m-miR-376b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM43
m-miR-376a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM43
m-miR-411
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM43
m-miR-380-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM43
hm-miR-412
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM26
hmr-miR-27b
6.46
8.12
7.01
7.72
6.00
6.48
6.42
6.00
6.00


EAM26
hmr-miR-26a
10.10
10.36
9.80
10.53
10.61
10.61
10.21
9.69
10.16


EAM26
hmr-miR-24
6.06
6.00
6.00
6.00
8.19
6.00
7.34
6.00
7.32


EAM26
hmr-miR-23b
8.76
8.71
8.23
9.80
9.82
8.30
9.41
7.07
9.69


EAM26
hmr-miR-23a
8.70
9.13
8.70
9.58
9.49
7.88
9.15
7.55
9.76


EAM25
h-miR-220
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.11
6.00


EAM25
hmr-miR-22
6.00
6.00
6.00
6.00
6.00
6.00
6.82
6.00
6.00


EAM24
hmr-miR-213
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM24
hmr-miR-21
8.75
9.03
10.40
10.10
6.00
9.43
9.46
8.42
9.73


EAM24
hmr-miR-20a
11.01
11.25
10.42
10.12
8.35
10.58
9.97
10.81
10.61


EAM23
hmr-miR-19b
7.94
9.23
8.64
6.80
7.05
6.00
8.35
8.53
7.39


EAM23
hmr-miR-196a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM
hm-miR-148a
6.00
6.00
6.00
6.00
6.00
6.05
6.00
6.00
6.00


EAM21
hmr-miR-145
6.00
6.16
6.00
7.14
6.00
6.00
6.00
6.00
6.00


EAM21
hmr-miR-144
7.20
6.00
6.00
7.46
6.88
6.00
6.17
9.44
7.76


EAM
hmr-miR-143
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM38
r-miR-346
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM39
r-miR-347
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM39
r-miR-349
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA223
hmr-miR-33
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM27
hmr-miR-96
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM27
hmr-miR-9
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM27
hmr-miR-30d
7.77
7.93
7.87
7.45
7.53
6.00
8.04
7.48
6.00


EAM28
mr-miR-10b
7.30
7.23
6.00
6.00
6.00
7.97
6.00
6.00
7.89


EAM29
hm-miR-188
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM29
hmr-miR-193a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM30
h-miR-198
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM23
hmr-miR-192
6.00
6.00
6.00
6.00
6.00
6.00
6.00
8.50
6.00


EAM23
hmr-miR-187
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM23
hmr-miR-183
6.00
6.00
6.00
6.00
6.00
6.00
6.00
8.16
6.00


EAM22
hm-miR-182
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.48


EAM
hmr-miR-154
6.00
6.00
6.00
6.39
6.00
6.00
6.00
6.00
6.00


EAM
hmr-miR-153
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM
hmr-miR-152
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM21
hmr-miR-150
11.16
6.00
9.51
11.36
12.16
11.59
11.15
9.72
10.24


EAM
hm-miR-149
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM
hmr-miR-148b
6.00
6.00
6.00
6.00
6.00
7.27
6.00
6.00
6.00


EAM
hmr-miR-30c
8.38
8.74
9.34
8.39
8.71
9.24
9.92
6.00
9.42


EAM26
hmr-miR-29a
8.05
6.00
6.00
6.00
7.29
9.38
8.34
6.29
6.00


EAM30
hmr-miR-200b
6.00
6.23
6.00
6.00
6.00
6.51
6.00
6.00
6.00


EAM30
hm-miR-199a*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM30
h-miR-197
7.57
6.00
6.61
8.15
10.15
6.98
9.26
7.21
8.36


EAM29
hmr-miR-195
8.90
8.67
10.18
9.61
9.73
9.28
8.75
10.26
9.42


JLA91
hmr-miR-99b
6.00
6.00
9.02
6.00
6.00
6.00
7.63
9.71
6.00


JLA92
hmr-miR-433
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA93
hmr-miR-431
6.00
6.00
6.00
7.98
6.00
6.00
6.00
6.00
6.00


JLA94
hmr-miR-365
6.77
6.53
6.00
9.78
6.00
6.79
6.00
6.00
9.08


JLA95
hmr-miR-450
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA96
hmr-miR-449
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA99
hmr-miR-448
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA103
hmr-miR-424
7.35
7.34
6.00
6.00
7.82
6.00
6.20
6.00
7.17


JLA105
hm-miR-361
6.00
6.98
7.83
6.00
6.00
6.23
7.28
6.00
6.00


JLA106
hm-miR-375
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA107
hm-miR-377
6.00
6.78
6.00
6.00
6.00
6.00
6.19
6.00
6.00


JLA108
hm-miR-378
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA109
hm-miR-379
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA110
hm-miR-380-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA111
hm-miR-381
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA112
hm-miR-382
6.00
6.00
6.00
7.52
6.00
6.00
7.61
6.00
6.00


JLA115
hm-miR-384
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA116
hm-miR-425
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA117
hm-miR-452
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA118
hm-miR-30e-3p
6.00
6.00
6.00
6.00
6.00
6.36
6.00
6.00
6.00


JLA104
mr-miR-129-3p
8.05
6.65
6.00
10.17
6.00
6.00
6.00
9.32
7.47


JLA98
mr-miR-429
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA101
mr-miR-330
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA102
mr-miR-322
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA114
m-miR-383
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA5
hmr-miR-451
8.40
6.00
6.13
7.43
9.12
9.14
7.74
7.56
7.28


JLA201
r-miR-421
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA202
m-miR-463
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA203
m-miR-464
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA204
m-miR-465
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA205
m-miR-466
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA206
m-miR-467
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA207
m-miR-468
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA208
m-miR-469
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA209
m-miR-470
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA210
m-miR-471
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM325
hmr-miR-27a
7.10
8.39
6.53
8.76
6.00
6.63
7.02
6.00
6.84


EAM326
hmr-miR-296
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM327
hmr-miR-299-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM328
hmr-miR-301
6.00
6.00
6.00
6.00
6.00
6.09
6.00
6.00
6.00


EAM329
hm-miR-302a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM330
hmr-miR-30a-5p
6.00
6.00
6.00
6.00
6.00
6.29
6.00
6.00
6.00


EAM331
hmr-miR-30e-5p
6.00
7.15
6.00
6.00
6.00
7.02
6.00
6.00
6.00


EAM332
hmr-miR-31
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM333
hmr-miR-32
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM335
h-miR-34b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM336
hmr-miR-34c
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM337
hmr-miR-93
8.55
9.94
9.41
7.91
6.00
9.76
9.69
8.17
8.93


EAM208
hmr-miR-141
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM207
hmr-miR-140
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA222
hmr-miR-139
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA220
hmr-miR-138
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM203
hmr-miR-135a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM200
hmr-miR-133a
6.00
6.00
6.00
6.99
6.00
6.00
6.00
6.00
6.00


EAM195
hmr-miR-128b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM194
hmr-miR-128a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM254
hmr-miR-219
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM257
hmr-miR-221
7.37
7.46
6.00
8.97
8.93
6.00
6.00
6.00
6.00


EAM258
hmr-miR-222
8.32
7.76
6.95
8.78
6.00
6.00
6.83
6.00
6.00


EAM259
hmr-miR-223
8.60
8.36
9.22
9.19
8.58
8.02
8.85
6.64
9.70


JLA211
m-miR-434-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA212
m-miR-434-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA213
m-miR-433-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA2
hsa-miR-522
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA3
hsa-miR-495
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA200
r-miR-297
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA6
hsa-miR-518e
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA7
hsa-miR-519a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA8
hsa-mir-527*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA72
hmr-miR-140*
6.03
6.02
6.00
8.06
6.00
6.93
7.57
6.48
6.93


JLA10
hsa-miR-521
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA12
hsa-miR-362
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA74
hsa-mir-18*
6.00
6.00
6.00
6.39
6.00
6.00
6.00
6.00
6.00


JLA14
hm-miR-363
6.00
6.00
7.63
6.00
6.00
6.00
7.63
6.00
6.00


JLA77
hsa-mir-19b-1*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA17
hsa-mir-520c,b,f
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA79
hsa-mir-23a*
6.00
6.00
6.00
6.00
6.00
6.57
6.00
6.00
7.64


JLA20
hsa-miR-369-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA81
hsa-mir-339*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA23
hsa-mir-342*
6.00
6.81
6.00
8.42
7.43
6.00
6.00
7.48
6.00


JLA24
hsa-mir-19a*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA26
hsa-miR-517a,b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA27
hsa-miR-516-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA28
hsa-miR-518b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA29
hsa-miR-519d
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA73
hr-mir-151*
7.22
6.01
7.57
6.87
8.90
7.45
7.30
6.00
7.78


JLA31
hsa-mir-28*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA33
hsa-mir-519a-2*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA34
hsa-mir-26b*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA35
hsa-miR-526c
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA36
hsa-miR-527
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA38
hsa-mir-29b-2*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA39
hsa-let-7g*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA40
hsa-miR-518a
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA41
hsa-miR-523
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA44
hsa-miR-515-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA45
hsa-mir-146b*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA49
hsa-mir-222*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA53
hsa-mir-24*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA55
hsa-miR-503
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA57
hsa-mir-505
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA82
hsa-mir-423*
10.16
9.82
9.90
11.38
12.04
10.39
11.13
9.84
11.00


JLA66
hsa-miR-432
6.00
7.28
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA83
hsa-mir-425*
7.93
7.46
6.00
6.51
6.00
6.89
7.58
6.00
8.04


JLA84
hsa-mir-92-1*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA69
hsa-mir-193*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA70
hsa-miR-515-5p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA71
hsa-mir-516-1*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA85
hsa-mir-30d*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA125
h-miR-20b
9.71
9.91
8.67
8.72
8.40
8.95
9.35
10.04
9.35


JLA198
h-miR-191*
6.00
6.00
6.74
6.36
6.15
6.00
6.00
7.19
6.00


JLA199
h-miR-154*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM316
h-miR-147
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM317
h-miR-155
8.85
9.85
8.50
6.00
6.00
8.82
7.46
6.00
8.90


EAM318
h-miR-17-3p
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA195
h-miR-200a*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA196
h-miR-302a*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA197
h-miR-299-3p
6.00
6.00
6.00
6.23
6.00
6.00
6.00
6.00
7.60


EAM319
h-miR-182*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM405
h-miR-302b
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.19


EAM406
h-miR-302b*
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


EAM392
r-miR-352
7.72
6.58
7.71
8.32
7.78
8.00
8.07
8.34
8.37


JLA123
h-miR-423
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00


JLA124
h-miR-18b
7.19
6.00
6.00
6.00
6.00
6.00
6.00
6.00
6.00









Data were normalized, log 2-transformed and thresholded at 6. Readings for samples are in columns and readings for miRNAs are in rows. Due to page limitation, every page lists only a subset of samples and miRNAs. The data will also be available online.

## Claims

1. A method of treating thrombocytopenia in a host in need thereof, the method comprising administering an effective amount of an agent that increases miR-150 expression in a cell to a host, wherein the agent comprises a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1.

2. The method of claim 1, wherein the cell is a progenitor cell.

3. The method of claim 2, wherein the progenitor cell is a hematopoietic progenitor cell.

4. The method of claim 1, wherein the agent is a vector comprising the nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1.

5. The method of claim 4, wherein the vector is a virus.

6. A method of treating thrombocytopenia in a host in need thereof, the method comprising:
a. obtaining a sample of hematopoietic progenitor cells from said host;
b. contacting the hematopoietic progenitor cells with an agent comprising a nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1; and
c. introducing the cell from step b into the host.

7. The method of claim 6, wherein the agent is a vector comprising the nucleic acid sequence that is at least 90% identical to SEQ. ID. No. 1.