# Patent

## Title
Methods, compositions, and kits comprising linker probes for quantifying polynucleotides

## Abstract

The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3′ target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3′ target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 13/612,485, filed Sep. 12, 2012, which is a continuation of U.S. application Ser. No. 12/543,466, filed Aug. 18, 2009, now U.S. Pat. No. 9,068,222, which is a continuation of U.S. application Ser. No. 10/947,460, filed Sep. 21, 2004, now U.S. Pat. No. 7,575,863, which claims the benefit of U.S. Provisional Application 60/575,661, filed May 28, 2004, each of which is incorporated herein by reference in its entirety.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 6, 2009, is named 533USC1.txt, and is 118,670 bytes in size.
FIELD
The present teachings are in the field of molecular and cell biology, specifically in the field of detecting target polynucleotides such as miRNA.
INTRODUCTION
RNA interference (RNAi) is a highly coordinated, sequence-specific mechanism involved in posttranscriptional gene regulation. During the initial steps of process, a ribonuclease (RNase) II-like enzyme called Dicer reduces long double-strand RNA (dsRNA) and complex hairpin precursors into: 1) small interfering RNAs (siRNA) that degrade messenger RNA (mRNA) and 2) micro RNAs (miRNAs) that can target mRNAs for cleavage or attenuate translation.
The siRNA class of molecules is thought to be comprised of 21-23 nucleotide (nt) depluxes with characteristic dinucleotide 3′ overhangs (Ambros et al., 2003, RNA, 9 (3), 277-279). siRNA has been shown to act as the functional intermediate in RNAi, specifically directing cleavage of complementary mRNA targets in a process that is commonly regarded to be an antiviral cellular defense mechanism (Elbashir et al., 2001, Nature, 411:6836), 494-498, Elbashir et al., 2001, Genes and Development, 15 (2), 188-200). Target RNA cleavage is catalyzed by the RNA-induced silencing complex (RISC), which functions as a siRNA directed endonuclease (reviewed in Bartel, 2004, Cell, 116 (2), 281-297).
Micro RNAs (miRNAs) typically comprise single-stranded, endogenous oligoribonucleotides of roughly 22 (18-25) bases in length that are processed from larger stem-looped precursor RNAs. The first genes recognized to encode miRNAs, lin-4 and let-7 of C. elegans, were identified on the basis of the developmental timing defects associated with the loss-of-function mutations (Lee et al., 1993, Cell, 75 (5), 843-854; Reinhart et al., 2000, Nature, 403, (6772), 901-906; reviewed by Pasquinelli et al., 2002, Annual Review of Cell and Developmental Biology, 18, 495-513). The breadth and importance of miRNA-directed gene regulation are coming into focus as more miRNAs and regulatory targets and functions are discovered. To date, a total of at least 700 miRNAs have been identified in C. elegans, Drosophila (Fire et al., 1998, Nature, 391 (6669), 805-811), mouse, human (Lagos-Quintana et al., 2001, Science, 294 (5543), 853-858), and plants (Reinhart et al., 2002, Genes and Development, 16 (13), 1616-1626). Their sequences are typically conserved among different species. Size ranges from 18 to 25 nucleotides for miRNAs are the most commonly observed to date.
The function of most miRNAs is not known. Recently discovered miRNA functions include control of cell proliferation, cell death, and fat metabolism in flies (Brennecke et al., 2003, cell, 113 (1), 25-36; Xu et al, 2003, Current Biology, 13 (9), 790-795), neuronal patterning in nematodes (Johnston and Hobert, 2003, Nature, 426 (6968), 845-849), modulation of hematopoietic lineage differentiation in mammals (Chen et al., 2004, Science, 303 (5654), 83-87), and control of leaf and flower development in plants (Aukerman and Sakai, 2003, Plant Cell, 15 (11), 2730-2741; Chen, 2003, Science, 303 (5666):2022-2025; Emery et al., 2003, Current Biology, 13 (20), 1768-1774; Palatnik et al., 2003, Nature, 425 (6955), 257-263). There is speculation that miRNAs may represent a new aspect of gene regulation.
Most miRNAs have been discovered by cloning. There are few cloning kits available for researchers from Ambion and QIAGEN etc. The process is laborious and less accurate. Further, there has been little reliable technology available for miRNA quantitation (Allawi et al., Third Wave Technologies, RNA. 2004 July; 10(7):1153-61). Northern blotting has been used but results are not quantitative (Lagos-Quitana et al., 2001, Science, 294 (5543), 853-854). Many miRNA researchers are interested in monitoring the level of the miRNAs at different tissues, at the different stages of development, or after treatment with various chemical agents. However, the short length of miRNAs has their study difficult.
SUMMARY
In some embodiments, the present teachings provide a method for detecting a micro RNA (miRNA) comprising; hybridizing the miRNA and a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target-specific portion, wherein the 3′ target-specific portion base pairs with the 3′ end region of the miRNA; extending the linker probe to form an extension reaction product; amplifying the extension reaction product to form an amplification product; and, detecting the miRNA.
In some embodiments, the present teachings provide a method for detecting a target polynucleotide comprising; hybridizing the target polynucleotide and a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target-specific portion, wherein the 3′ target-specific portion base pairs with the 3′ end region of the target polynucleotide; extending the linker probe to form an extension reaction product; amplifying the extension reaction product to form an amplification product in the presence of a detector probe, wherein the detector probe comprises a nucleotide of the linker probe stem in the amplification product or a nucleotide of the linker probe stem complement in the amplification product; and, detecting the target polynucleotide.
In some embodiments, the present teachings provide a method for detecting a miRNA molecule comprising; hybridizing the miRNA molecule and a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target specific portion, wherein the 3′ target-specific portion base pairs with the 3′ end region of the target polynucleotide; extending the linker probe to form an extension reaction product; amplifying the extension reaction product in the presence of a detector probe to form an amplification product, wherein the detector probe comprises a nucleotide of the linker probe stem in the amplification product or a nucleotide of the linker probe stem complement in the amplification product, and the detector probe further comprises a nucleotide of the 3′ end region of the miRNA in the amplification product or a nucleotide of the 3′ end region of the miRNA complement in the amplification product; and, detecting the miRNA molecule.
In some embodiments, the present teachings provide a method for detecting two different miRNAs from a single hybridization reaction comprising; hybridizing a first miRNA and a first linker probe, and a second miRNA and a second linker probe, wherein the first linker probe and the second linker probe each comprise a loop, a stem, and a 3′ target-specific portion, wherein the 3′ target-specific portion of the first linker probe base pairs with the 3′ end region of the first miRNA, and wherein the 3′ target-specific portion of the second linker probe base pairs with the 3′ end region of the second miRNA; extending the first linker probe and the second linker probe to form extension reaction products; dividing the extension reaction products into a first amplification reaction to form a first amplification reaction product, and a second amplification reaction to form a second amplification reaction product, wherein a primer in the first amplification reaction corresponds with the first miRNA and not the second miRNA, and a primer in the second amplification reaction corresponds with the second miRNA and not the first miRNA, wherein a first detector probe in the first amplification reaction differs from a second detector probe in the second amplification reaction, wherein the first detector probe comprises a nucleotide of the first linker probe stem of the amplification product or a nucleotide of the first linker probe stem complement in the first amplification product, wherein the second detector probe comprises a nucleotide of the second linker probe stem of the amplification product or a nucleotide of the second linker probe stem complement in the amplification product; and, detecting the two different miRNAs.
In some embodiments, the present teachings provide a method for detecting two different target polynucleotides from a single hybridization reaction comprising; hybridizing a first target polynucleotide and a first linker probe, and a second target polynucleotide and a second linker probe, wherein the first linker probe and the second linker probe each comprise a loop, a stem, and a 3′ target-specific portion, wherein the 3′ target-specific portion of the first linker probe base pairs with the 3′ end region of the first target polynucleotide, and wherein the 3′ target-specific portion of the second linker probe base pairs with the 3′ end region of the second target polynucleotide; extending the first linker probe and the second linker probe to form extension reaction products; dividing the extension reaction products into a first amplification reaction to form a first amplification reaction product and a second amplification reaction to form a second amplification reaction product; and, detecting the two different miRNA molecules.
In some embodiments, the present teachings provide a method for detecting a miRNA molecule from a cell lysate comprising; hybridizing the miRNA molecule from the cell lysate with a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target specific portion, wherein the 3′ target-specific portion base pairs with the 3′ end region of the miRNA; extending the linker probe to form an extension reaction product; amplifying the extension reaction product to form an amplification product in the presence of a detector probe, wherein the detector probe comprises a nucleotide of the linker probe stem of the amplification product or a nucleotide of the linker probe stem complement in the amplification product, and the detector probe further comprises a nucleotide of the 3′ end region of the miRNA in the amplification product or a nucleotide of the 3′ end region of the miRNA complement in the amplification product; and, detecting the miRNA molecule.
A kit comprising; a reverse transcriptase and a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target-specific portion, wherein the 3′ target-specific portion corresponds to a miRNA.
The present teachings contemplate method for detecting a miRNA molecule comprising a step of hybridizing, a step of extending, a step of amplifying, and a step of detecting.
These and other features of the present teachings are set forth herein.



BRIEF DESCRIPTION OF THE DRAWINGS
The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
FIGS. 1A, 1B, and 1C depict certain aspects of various compositions according to some embodiments of the present teachings.
FIGS. 2A, 2B, 2C, and 2D depict certain aspects of various compositions according to some embodiments of the present teachings.
FIG. 3 depicts certain sequences of various compositions according to some embodiments of the present teachings. FIG. 3 depicts SEQ ID No. 780, the oligonucleotide for the micro RNA MiR-16 (boxed, 11) and a linker probe (13).
FIG. 4 depicts one single-plex assay design according to some embodiments of the present teachings.
FIG. 5 depicts an overview of a multiplex assay design according to some embodiments of the present teachings.
FIG. 6 depicts a multiplex assay design according to some embodiments of the present teachings.



DESCRIPTION OF EXEMPLARY EMBODIMENTS
Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way. The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control. It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, etc discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein. In this application, the use of the singular includes the plural unless specifically stated otherwise. For example, “a primer” means that more than one primer can, but need not, be present; for example but without limitation, one or more copies of a particular primer species, as well as one or more versions of a particular primer type, for example but not limited to, a multiplicity of different forward primers. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.
SOME DEFINITIONS
As used herein, the term “target polynucleotide” refers to a polynucleotide sequence that is sought to be detected. The target polynucleotide can be obtained from any source, and can comprise any number of different compositional components. For example, the target can be nucleic acid (e.g. DNA or RNA), transfer RNA, sRNA, and can comprise nucleic acid analogs or other nucleic acid mimic. The target can be methylated, non-methylated, or both. The target can be bisulfite-treated and non-methylated cytosines converted to uracil. Further, it will be appreciated that “target polynucleotide” can refer to the target polynucleotide itself, as well as surrogates thereof, for example amplification products, and native sequences. In some embodiments, the target polynucleotide is a miRNA molecule. In some embodiments, the target polynucleotide lacks a poly-A tail. In some embodiments, the target polynucleotide is a short DNA molecule derived from a degraded source, such as can be found in for example but not limited to forensics samples (see for example Butler, 2001, Forensic DNA Typing: Biology and Technology Behind STR Markers. The target polynucleotides of the present teachings can be derived from any of a number of sources, including without limitation, viruses, prokaryotes, eukaryotes, for example but not limited to plants, fungi, and animals. These sources may include, but are not limited to, whole blood, a tissue biopsy, lymph, bone marrow, amniotic fluid, hair, skin, semen, biowarfare agents, anal secretions, vaginal secretions, perspiration, saliva, buccal swabs, various environmental samples (for example, agricultural, water, and soil), research samples generally, purified samples generally, cultured cells, and lysed cells. It will be appreciated that target polynucleotides can be isolated from samples using any of a variety of procedures known in the art, for example the Applied Biosystems ABI PrismTM 6100 Nucleic Acid PrepStation, and the ABI PrismTM 6700 Automated Nucleic Acid Workstation, Boom et al., U.S. Pat. No. 5,234,809, mirVana RNA isolation kit (Ambion), etc. It will be appreciated that target polynucleotides can be cut or sheared prior to analysis, including the use of such procedures as mechanical force, sonication, restriction endonuclease cleavage, or any method known in the art. In general, the target polynucleotides of the present teachings will be single stranded, though in some embodiments the target polynucleotide can be double stranded, and a single strand can result from denaturation.
As used herein, the term “3′ end region of the target polynucleotide” refers to the region of the target to which the 3′ target specific portion of the linker probe hybridizes. In some embodiments there can be a gap between the 3′ end region of the target polynucleotide and the 5′ end of the linker probe, with extension reactions filling in the gap, though generally such scenarios are not preferred because of the likely destabilizing effects on the duplex. In some embodiments, a miRNA molecule is the target, in which case the term “3′ end region of the miRNA” is used.
As used herein, the term “linker probe” refers to a molecule comprising a 3′ target specific portion, a stem, and a loop. Illustrative linker probes are depicted in FIGS. 2A-2D and elsewhere in the present teachings. It will be appreciated that the linker probes, as well as the primers of the present teachings, can be comprised of ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar-backbone oligonucleotides, nucleotide analogs, or combinations thereof. For some illustrative teachings of various nucleotide analogs etc, see Fasman, 1989, Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla., Loakes, N. A. R. 2001, vol 29:2437-2447, and Pellestor et al., Int J Mol Med. 2004 April; 13(4):521-5.), references cited therein, and recent articles citing these reviews. It will be appreciated that the selection of the linker probes to query a given target polynucleotide sequence, and the selection of which collection of target polynucleotide sequences to query in a given reaction with which collection of linker probes, will involve procedures generally known in the art, and can involve the use of algorithms to select for those sequences with minimal secondary and tertiary structure, those targets with minimal sequence redundancy with other regions of the genome, those target regions with desirable thermodynamic characteristics, and other parameters desirable for the context at hand.
As used herein, the term “3′ target-specific portion” refers to the single stranded portion of a linker probe that is complementary to a target polynucleotide. The 3′ target-specific portion is located downstream from the stem of the linker probe. Generally, the 3′ target-specific portion is between 6 and 8 nucleotides long. In some embodiments, the 3′ target-specific portion is 7 nucleotides long. It will be appreciated that routine experimentation can produce other lengths, and that 3′ target-specific portions that are longer than 8 nucleotides or shorter than 6 nucleotides are also contemplated by the present teachings. Generally, the 3′-most nucleotides of the 3′ target-specific portion should have minimal complementarity overlap, or no overlap at all, with the 3′ nucleotides of the forward primer; it will be appreciated that overlap in these regions can produce undesired primer dimer amplification products in subsequent amplification reactions. In some embodiments, the overlap between the 3′-most nucleotides of the 3′ target-specific portion and the 3′ nucleotides of the forward primer is 0, 1, 2, or 3 nucleotides. In some embodiments, greater than 3 nucleotides can be complementary between the 3′-most nucleotides of the 3′ target-specific portion and the 3′ nucleotides of the forward primer, but generally such scenarios will be accompanied by additional non-complementary nucleotides interspersed therein. In some embodiments, modified bases such as LNA can be used in the 3′ target specific portion to increase the Tm of the linker probe (see for example Petersen et al., Trends in Biochemistry (2003), 21:2:74-81). In some embodiments, universal bases can be used, for example to allow for smaller libraries of linker probes. Universal bases can also be used in the 3′ target specific portion to allow for the detection of unknown targets. For some descriptions of universal bases, see for example Loakes et al., Nucleic Acids Research, 2001, Volume 29, No. 12, 2437-2447. In some embodiments, modifications including but not limited to LNAs and universal bases can improve reverse transcription specificity and potentially enhance detection specificity.
As used herein, the term “stem” refers to the double stranded region of the linker probe that is between the 3′ target-specific portion and the loop. Generally, the stem is between 6 and 20 nucleotides long (that is, 6-20 complementary pairs of nucleotides, for a total of 12-40 distinct nucleotides). In some embodiments, the stem is 8-14 nucleotides long. As a general matter, in those embodiments in which a portion of the detector probe is encoded in the stem, the stem can be longer. In those embodiments in which a portion of the detector probe is not encoded in the stem, the stem can be shorter. Those in the art will appreciate that stems shorter that 6 nucleotides and longer than 20 nucleotides can be identified in the course of routine methodology and without undue experimentation, and that such shorter and longer stems are contemplated by the present teachings. In some embodiments, the stem can comprise an identifying portion.
As used herein, the term “loop” refers to a region of the linker probe that is located between the two complementary strands of the stem, as depicted in FIGS. 1A-1C and elsewhere in the present teachings. Typically, the loop comprises single stranded nucleotides, though other moieties modified DNA or RNA, Carbon spacers such as C18, and/or PEG (polyethylene glycol) are also possible. Generally, the loop is between 4 and 20 nucleotides long. In some embodiments, the loop is between 14 and 18 nucleotides long. In some embodiments, the loop is 16 nucleotides long. As a general matter, in those embodiments in which a reverse primer is encoded in the loop, the loop can generally be longer. In those embodiments in which the reverse primer corresponds to both the target polynucleotide as well as the loop, the loop can generally be shorter. Those in the art will appreciate that loops shorter that 4 nucleotides and longer than 20 nucleotides can be identified in the course of routine methodology and without undue experimentation, and that such shorter and longer loops are contemplated by the present teachings. In some embodiments, the loop can comprise an identifying portion.
As used herein, the term “identifying portion” refers to a moiety or moieties that can be used to identify a particular linker probe species, and as a result determine a target polynucleotide sequence, and can refer to a variety of distinguishable moieties including zipcodes, a known number of nucleobases, and combinations thereof. In some embodiments, an identifying portion, or an identifying portion complement, can hybridize to a detector probe, thereby allowing detection of a target polynucleotide sequence in a decoding reaction. The terms “identifying portion complement” typically refers to at least one oligonucleotide that comprises at least one sequence of nucleobases that are at least substantially complementary to and hybridize with their corresponding identifying portion. In some embodiments, identifying portion complements serve as capture moieties for attaching at least one identifier portion:element complex to at least one substrate; serve as “pull-out” sequences for bulk separation procedures; or both as capture moieties and as pull-out sequences (see for example O'Neil, et al., U.S. Pat. Nos. 6,638,760, 6,514,699, 6,146,511, and 6,124,092). Typically, identifying portions and their corresponding identifying portion complements are selected to minimize: internal, self-hybridization; cross-hybridization with different identifying portion species, nucleotide sequences in a reaction composition, including but not limited to gDNA, different species of identifying portion complements, or target-specific portions of probes, and the like; but should be amenable to facile hybridization between the identifying portion and its corresponding identifying portion complement. Identifying portion sequences and identifying portion complement sequences can be selected by any suitable method, for example but not limited to, computer algorithms such as described in PCT Publication Nos. WO 96/12014 and WO 96/41011 and in European Publication No. EP 799,897; and the algorithm and parameters of SantaLucia (Proc. Natl. Acad. Sci. 95:1460-65 (1998)). Descriptions of identifying portions can be found in, among other places, U.S. Pat. No. 6,309,829 (referred to as “tag segment” therein); U.S. Pat. No. 6,451,525 (referred to as “tag segment” therein); U.S. Pat. No. 6,309,829 (referred to as “tag segment” therein); U.S. Pat. No. 5,981,176 (referred to as “grid oligonucleotides” therein); U.S. Pat. No. 5,935,793 (referred to as “identifier tags” therein); and PCT Publication No. WO 01/92579 (referred to as “addressable support-specific sequences” therein). In some embodiments, the stem of the linker probe, the loop of the linker probe, or combinations thereof can comprise an identifying portion, and the detector probe can hybridize to the corresponding identifying portion. In some embodiments, the detector probe can hybridize to both the identifying portion as well as sequence corresponding to the target polynucleotide. In some embodiments, at least two identifying portion: identifying portion complement duplexes have melting temperatures that fall within a ΔTm range (Tmax−Tmin) of no more than 10° C. of each other. In some embodiments, at least two identifying portion: identifying portion complement duplexes have melting temperatures that fall within a ΔTm range of 5° C. or less of each other. In some embodiments, at least two identifying portion: identifying portion complement duplexes have melting temperatures that fall within a ΔTm range of 2° C. or less of each other. In some embodiments, at least one identifying portion or at least one identifying portion complement is used to separate the element to which it is bound from at least one component of a ligation reaction composition, a digestion reaction composition, an amplified ligation reaction composition, or the like. In some embodiments, identifying portions are used to attach at least one ligation product, at least one ligation product surrogate, or combinations thereof, to at least one substrate. In some embodiments, at least one ligation product, at least one ligation product surrogate, or combinations thereof, comprise the same identifying portion. Examples of separation approaches include but are not limited to, separating a multiplicity of different element: identifying portion species using the same identifying portion complement, tethering a multiplicity of different element: identifying portion species to a substrate comprising the same identifying portion complement, or both. In some embodiments, at least one identifying portion complement comprises at least one label, at least one mobility modifier, at least one label binding portion, or combinations thereof. In some embodiments, at least one identifying portion complement is annealed to at least one corresponding identifying portion and, subsequently, at least part of that identifying portion complement is released and detected, see for example Published P.C.T. Application WO04/4634 to Rosenblum et al., and Published P.C.T. Application WO01/92579 to Wenz et al.,
As used herein, the term “extension reaction” refers to an elongation reaction in which the 3′ target specific portion of a linker probe is extended to form an extension reaction product comprising a strand complementary to the target polynucleotide. In some embodiments, the target polynucleotide is a miRNA molecule and the extension reaction is a reverse transcription reaction comprising a reverse transcriptase. In some embodiments, the extension reaction is a reverse transcription reaction comprising a polymerase derived from a Eubacteria. In some embodiments, the extension reaction can comprise rTth polymerase, for example as commercially available from Applied Biosystems catalog number N808-0192, and N808-0098. In some embodiments, the target polynucleotide is a miRNA or other RNA molecule, and as such it will be appreciated that the use of polymerases that also comprise reverse transcription properties can allow for some embodiments of the present teachings to comprise a first reverse transcription reaction followed thereafter by an amplification reaction, thereby allowing for the consolidation of two reactions in essentially a single reaction. In some embodiments, the target polynucleotide is a short DNA molecule and the extension reaction comprises a polymerase and results in the synthesis of a 2nd strand of DNA. In some embodiments, the consolidation of the extension reaction and a subsequent amplification reaction is further contemplated by the present teachings.
As used herein, the term “primer portion” refers to a region of a polynucleotide sequence that can serve directly, or by virtue of its complement, as the template upon which a primer can anneal for any of a variety of primer nucleotide extension reactions known in the art (for example, PCR). It will be appreciated by those of skill in the art that when two primer portions are present on a single polynucleotide, the orientation of the two primer portions is generally different. For example, one PCR primer can directly hybridize to a first primer portion, while the other PCR primer can hybridize to the complement of the second primer portion. In addition, “universal” primers and primer portions as used herein are generally chosen to be as unique as possible given the particular assays and host genomes to ensure specificity of the assay.
As used herein, the term “forward primer” refers to a primer that comprises an extension reaction product portion and a tail portion. The extension reaction product portion of the forward primer hybridizes to the extension reaction product. Generally, the extension reaction product portion of the forward primer is between 9 and 19 nucleotides in length. In some embodiments, the extension reaction product portion of the forward primer is 16 nucleotides. The tail portion is located upstream from the extension reaction product portion, and is not complementary with the extension reaction product; after a round of amplification however, the tail portion can hybridize to complementary sequence of amplification products. Generally, the tail portion of the forward primer is between 5-8 nucleotides long. In some embodiments, the tail portion of the forward primer is 6 nucleotides long. Those in the art will appreciate that forward primer tail portion lengths shorter than 5 nucleotides and longer than 8 nucleotides can be identified in the course of routine methodology and without undue experimentation, and that such shorter and longer forward primer tail portion lengths are contemplated by the present teachings. Further, those in the art will appreciate that lengths of the extension reaction product portion of the forward primer shorter than 9 nucleotides in length and longer than 19 nucleotides in length can be identified in the course of routine methodology and without undue experimentation, and that such shorter and longer extension reaction product portion of forward primers are contemplated by the present teachings.
As used herein, the term “reverse primer” refers to a primer that when extended forms a strand complementary to the target polynucleotide. In some embodiments, the reverse primer corresponds with a region of the loop of the linker probe. Following the extension reaction, the forward primer can be extended to form a second strand product. The reverse primer hybridizes with this second strand product, and can be extended to continue the amplification reaction. In some embodiments, the reverse primer corresponds with a region of the loop of the linker probe, a region of the stem of the linker probe, a region of the target polynucleotide, or combinations thereof. Generally, the reverse primer is between 13-16 nucleotides long. In some embodiments the reverse primer is 14 nucleotides long. In some embodiments, the reverse primer can further comprise a non-complementary tail region, though such a tail is not required. In some embodiments, the reverse primer is a “universal reverse primer,” which indicates that the sequence of the reverse primer can be used in a plurality of different reactions querying different target polynucleotides, but that the reverse primer nonetheless is the same sequence.
The term “upstream” as used herein takes on its customary meaning in molecular biology, and refers to the location of a region of a polynucleotide that is on the 5′ side of a “downstream” region. Correspondingly, the term “downstream” refers to the location of a region of a polynucleotide that is on the 3′ side of an “upstream” region.
As used herein, the term “hybridization” refers to the complementary base-pairing interaction of one nucleic acid with another nucleic acid that results in formation of a duplex, triplex, or other higher-ordered structure, and is used herein interchangeably with “annealing.” Typically, the primary interaction is base specific, e.g., A/T and G/C, by Watson/Crick and Hoogsteen-type hydrogen bonding. Base-stacking and hydrophobic interactions can also contribute to duplex stability. Conditions for hybridizing detector probes and primers to complementary and substantially complementary target sequences are well known, e.g., as described in Nucleic Acid Hybridization, A Practical Approach, B. Hames and S. Higgins, eds., IRL Press, Washington, D.C. (1985) and J. Wetmur and N. Davidson, Mol. Biol. 31:349 et seq. (1968). In general, whether such annealing takes place is influenced by, among other things, the length of the polynucleotides and the complementary, the pH, the temperature, the presence of mono- and divalent cations, the proportion of G and C nucleotides in the hybridizing region, the viscosity of the medium, and the presence of denaturants. Such variables influence the time required for hybridization. Thus, the preferred annealing conditions will depend upon the particular application. Such conditions, however, can be routinely determined by the person of ordinary skill in the art without undue experimentation. It will be appreciated that complementarity need not be perfect; there can be a small number of base pair mismatches that will minimally interfere with hybridization between the target sequence and the single stranded nucleic acids of the present teachings. However, if the number of base pair mismatches is so great that no hybridization can occur under minimally stringent conditions then the sequence is generally not a complementary target sequence. Thus, complementarity herein is meant that the probes or primers are sufficiently complementary to the target sequence to hybridize under the selected reaction conditions to achieve the ends of the present teachings.
As used herein, the term “amplifying” refers to any means by which at least a part of a target polynucleotide, target polynucleotide surrogate, or combinations thereof, is reproduced, typically in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Exemplary means for performing an amplifying step include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Q-replicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two-step multiplexed amplifications, rolling circle amplification (RCA) and the like, including multiplex versions or combinations thereof, for example but not limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also known as combined chain reaction—CCR), and the like. Descriptions of such techniques can be found in, among other places, Sambrook et al. Molecular Cloning, 3rd Edition; Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002), Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson et al., Curr Opin Biotechnol. 1993 February; 4(1):41-7, U.S. Pat. No. 6,027,998; U.S. Pat. No. 6,605,451, Barany et al., PCT Publication No. WO 97/31256; Wenz et al., PCT Publication No. WO 01/92579; Day et al., Genomics, 29(1): 152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the world wide web at: promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18-(2002); Lage et al., Genome Res. 2003 February; 13(2):294-307, and Landegren et al., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 November; 2(6):542-8, Cook et al., J Microbiol Methods. 2003 May; 53(2):165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 February; 12(1):21-7, U.S. Pat. No. 5,830,711, U.S. Pat. No. 6,027,889, U.S. Pat. No. 5,686,243, Published P.C.T. Application WO0056927A3, and Published P.C.T. Application WO9803673A1. In some embodiments, newly-formed nucleic acid duplexes are not initially denatured, but are used in their double-stranded form in one or more subsequent steps. An extension reaction is an amplifying technique that comprises elongating a linker probe that is annealed to a template in the 5′ to 3′ direction using an amplifying means such as a polymerase and/or reverse transcriptase. According to some embodiments, with appropriate buffers, salts, pH, temperature, and nucleotide triphosphates, including analogs thereof, i.e., under appropriate conditions, a polymerase incorporates nucleotides complementary to the template strand starting at the 3′-end of an annealed linker probe, to generate a complementary strand. In some embodiments, the polymerase used for extension lacks or substantially lacks 5′ exonuclease activity. In some embodiments of the present teachings, unconventional nucleotide bases can be introduced into the amplification reaction products and the products treated by enzymatic (e.g., glycosylases) and/or physical-chemical means in order to render the product incapable of acting as a template for subsequent amplifications. In some embodiments, uracil can be included as a nucleobase in the reaction mixture, thereby allowing for subsequent reactions to decontaminate carryover of previous uracil-containing products by the use of uracil-N-glycosylase (see for example Published P.C.T. Application WO9201814A2). In some embodiments of the present teachings, any of a variety of techniques can be employed prior to amplification in order to facilitate amplification success, as described for example in Radstrom et al., Mol Biotechnol. 2004 February; 26(2):133-46. In some embodiments, amplification can be achieved in a self-contained integrated approach comprising sample preparation and detection, as described for example in U.S. Pat. Nos. 6,153,425 and 6,649,378. Reversibly modified enzymes, for example but not limited to those described in U.S. Pat. No. 5,773,258, are also within the scope of the disclosed teachings. The present teachings also contemplate various uracil-based decontamination strategies, wherein for example uracil can be incorporated into an amplification reaction, and subsequent carry-over products removed with various glycosylase treatments (see for example U.S. Pat. No. 5,536,649, and U.S. Provisional Application 60/584,682 to Andersen et al.,). Those in the art will understand that any protein with the desired enzymatic activity can be used in the disclosed methods and kits. Descriptions of DNA polymerases, including reverse transcriptases, uracil N-glycosylase, and the like, can be found in, among other places, Twyman, Advanced Molecular Biology, BIOS Scientific Publishers, 1999; Enzyme Resource Guide, rev. 092298, Promega, 1998; Sambrook and Russell; Sambrook et al.; Lehninger; PCR: The Basics; and Ausbel et al.
As used herein, the term “detector probe” refers to a molecule used in an amplification reaction, typically for quantitative or real-time PCR analysis, as well as end-point analysis. Such detector probes can be used to monitor the amplification of the target polynucleotide. In some embodiments, detector probes present in an amplification reaction are suitable for monitoring the amount of amplicon(s) produced as a function of time. Such detector probes include, but are not limited to, the 5′-exonuclease assay (TaqMan® probes described herein (see also U.S. Pat. No. 5,538,848) various stem-loop molecular beacons (see e.g., U.S. Pat. Nos. 6,103,476 and 5,925,517 and Tyagi and Kramer, 1996, Nature Biotechnology 14:303-308), stemless or linear beacons (see, e.g., WO 99/21881), PNA Molecular BeaconsTM (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, e.g., Kubista et al., 2001, SPIE 4264:53-58), non-FRET probes (see, e.g., U.S. Pat. No. 6,150,097), Sunrise®/Amplifluor® probes (U.S. Pat. No. 6,548,250), stem-loop and duplex ScorpionTM probes (Solinas et al., 2001, Nucleic Acids Research 29:E96 and U.S. Pat. No. 6,589,743), bulge loop probes (U.S. Pat. No. 6,590,091), pseudo knot probes (U.S. Pat. No. 6,589,250), cyclicons (U.S. Pat. No. 6,383,752), MGB EclipseTM probe (Epoch Biosciences), hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA) light-up probes, self-assembled nanoparticle probes, and ferrocene-modified probes described, for example, in U.S. Pat. No. 6,485,901; Mhlanga et al., 2001, Methods 25:463-471; Whitcombe et al., 1999, Nature Biotechnology. 17:804-807; Isacsson et al., 2000, Molecular Cell Probes. 14:321-328; Svanvik et al., 2000, Anal Biochem. 281:26-35; Wolffs et al., 2001, Biotechniques 766:769-771; Tsourkas et al., 2002, Nucleic Acids Research. 30:4208-4215; Riccelli et al., 2002, Nucleic Acids Research 30:4088-4093; Zhang et al., 2002 Shanghai. 34:329-332; Maxwell et al., 2002, J. Am. Chem. Soc. 124:9606-9612; Broude et al., 2002, Trends Biotechnol. 20:249-56; Huang et al., 2002, Chem Res. Toxicol. 15:118-126; and Yu et al., 2001, J. Am. Chem. Soc 14:11155-11161. Detector probes can also comprise quenchers, including without limitation black hole quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch). Detector probes can also comprise two probes, wherein for example a fluor is on one probe, and a quencher is on the other probe, wherein hybridization of the two probes together on a target quenches the signal, or wherein hybridization on the target alters the signal signature via a change in fluorescence. Detector probes can also comprise sulfonate derivatives of fluorescenin dyes with SO3 instead of the carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of CY 5 (commercially available for example from Amersham). In some embodiments, interchelating labels are used such as ethidium bromide, SYBR® Green I (Molecular Probes), and PicoGreen® (Molecular Probes), thereby allowing visualization in real-time, or end point, of an amplification product in the absence of a detector probe. In some embodiments, real-time visualization can comprise both an intercalating detector probe and a sequence-based detector probe can be employed. In some embodiments, the detector probe is at least partially quenched when not hybridized to a complementary sequence in the amplification reaction, and is at least partially unquenched when hybridized to a complementary sequence in the amplification reaction. In some embodiments, the detector probes of the present teachings have a Tm of 63-69 C, though it will be appreciated that guided by the present teachings routine experimentation can result in detector probes with other Tms. In some embodiments, probes can further comprise various modifications such as a minor groove binder (see for example U.S. Pat. No. 6,486,308) to further provide desirable thermodynamic characteristics. In some embodiments, detector probes can correspond to identifying portions or identifying portion complements.
The term “corresponding” as used herein refers to a specific relationship between the elements to which the term refers. Some non-limiting examples of corresponding include: a linker probe can correspond with a target polynucleotide, and vice versa. A forward primer can correspond with a target polynucleotide, and vice versa. A linker probe can correspond with a forward primer for a given target polynucleotide, and vice versa. The 3′ target-specific portion of the linker probe can correspond with the 3′ region of a target polynucleotide, and vice versa. A detector probe can correspond with a particular region of a target polynucleotide and vice versa. A detector probe can correspond with a particular identifying portion and vice versa. In some cases, the corresponding elements can be complementary. In some cases, the corresponding elements are not complementary to each other, but one element can be complementary to the complement of another element.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
As used herein, the term “reaction vessel” generally refers to any container in which a reaction can occur in accordance with the present teachings. In some embodiments, a reaction vessel can be an eppendorf tube, and other containers of the sort in common practice in modern molecular biology laboratories. In some embodiments, a reaction vessel can be a well in microtitre plate, a spot on a glass slide, or a well in an Applied Biosystems TaqMan Low Density Array for gene expression (formerly MicroCardTM). For example, a plurality of reaction vessels can reside on the same support. In some embodiments, lab-on-a-chip like devices, available for example from Caliper and Fluidgm, can provide for reaction vessels. In some embodiments, various microfluidic approaches as described in U.S. Provisional Application 60/545,674 to Wenz et al., can be employed. It will be recognized that a variety of reaction vessel are available in the art and within the scope of the present teachings.
As used herein, the term “detection” refers to any of a variety of ways of determining the presence and/or quantity and/or identity of a target polynucleoteide. In some embodiments employing a donor moiety and signal moiety, one may use certain energy-transfer fluorescent dyes. Certain nonlimiting exemplary pairs of donors (donor moieties) and acceptors (signal moieties) are illustrated, e.g., in U.S. Pat. Nos. 5,863,727; 5,800,996; and 5,945,526. Use of some combinations of a donor and an acceptor have been called FRET (Fluorescent Resonance Energy Transfer). In some embodiments, fluorophores that can be used as signaling probes include, but are not limited to, rhodamine, cyanine 3 (Cy 3), cyanine 5 (Cy 5), fluorescein, VicTM LizTM, TamraTM, 5-FamTM, 6-FamTM, and Texas Red (Molecular Probes). (VicTM LizTM, TamraTM, 5-FamTM, and 6-FamTM (all available from Applied Biosystems, Foster City, Calif.). In some embodiments, the amount of detector probe that gives a fluorescent signal in response to an excited light typically relates to the amount of nucleic acid produced in the amplification reaction. Thus, in some embodiments, the amount of fluorescent signal is related to the amount of product created in the amplification reaction. In such embodiments, one can therefore measure the amount of amplification product by measuring the intensity of the fluorescent signal from the fluorescent indicator. According to some embodiments, one can employ an internal standard to quantify the amplification product indicated by the fluorescent signal. See, e.g., U.S. Pat. No. 5,736,333. Devices have been developed that can perform a thermal cycling reaction with compositions containing a fluorescent indicator, emit a light beam of a specified wavelength, read the intensity of the fluorescent dye, and display the intensity of fluorescence after each cycle. Devices comprising a thermal cycler, light beam emitter, and a fluorescent signal detector, have been described, e.g., in U.S. Pat. Nos. 5,928,907; 6,015,674; and 6,174,670, and include, but are not limited to the ABI Prism® 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.), the ABI GeneAmp® 5700 Sequence Detection System (Applied Biosystems, Foster City, Calif.), the ABI GeneAmp® 7300 Sequence Detection System (Applied Biosystems, Foster City, Calif.), and the ABI GeneAmp® 7500 Sequence Detection System (Applied Biosystems). In some embodiments, each of these functions can be performed by separate devices. For example, if one employs a Q-beta replicase reaction for amplification, the reaction may not take place in a thermal cycler, but could include a light beam emitted at a specific wavelength, detection of the fluorescent signal, and calculation and display of the amount of amplification product. In some embodiments, combined thermal cycling and fluorescence detecting devices can be used for precise quantification of target nucleic acid sequences in samples. In some embodiments, fluorescent signals can be detected and displayed during and/or after one or more thermal cycles, thus permitting monitoring of amplification products as the reactions occur in “real time.” In some embodiments, one can use the amount of amplification product and number of amplification cycles to calculate how much of the target nucleic acid sequence was in the sample prior to amplification. In some embodiments, one could simply monitor the amount of amplification product after a predetermined number of cycles sufficient to indicate the presence of the target nucleic acid sequence in the sample. One skilled in the art can easily determine, for any given sample type, primer sequence, and reaction condition, how many cycles are sufficient to determine the presence of a given target polynucleotide. As used herein, determining the presence of a target can comprise identifying it, as well as optionally quantifying it. In some embodiments, the amplification products can be scored as positive or negative as soon as a given number of cycles is complete. In some embodiments, the results may be transmitted electronically directly to a database and tabulated. Thus, in some embodiments, large numbers of samples can be processed and analyzed with less time and labor when such an instrument is used. In some embodiments, different detector probes may distinguish between different target polynucleoteides. A non-limiting example of such a probe is a 5′-nuclease fluorescent probe, such as a TaqMan® probe molecule, wherein a fluorescent molecule is attached to a fluorescence-quenching molecule through an oligonucleotide link element. In some embodiments, the oligonucleotide link element of the 5′-nuclease fluorescent probe binds to a specific sequence of an identifying portion or its complement. In some embodiments, different 5′-nuclease fluorescent probes, each fluorescing at different wavelengths, can distinguish between different amplification products within the same amplification reaction. For example, in some embodiments, one could use two different 5′-nuclease fluorescent probes that fluoresce at two different wavelengths (WLA and WLB) and that are specific to two different stem regions of two different extension reaction products (A′ and B′, respectively). Amplification product A′ is formed if target nucleic acid sequence A is in the sample, and amplification product B′ is formed if target nucleic acid sequence B is in the sample. In some embodiments, amplification product A′ and/or B′ may form even if the appropriate target nucleic acid sequence is not in the sample, but such occurs to a measurably lesser extent than when the appropriate target nucleic acid sequence is in the sample. After amplification, one can determine which specific target nucleic acid sequences are present in the sample based on the wavelength of signal detected and their intensity. Thus, if an appropriate detectable signal value of only wavelength WLA is detected, one would know that the sample includes target nucleic acid sequence A, but not target nucleic acid sequence B. If an appropriate detectable signal value of both wavelengths WLA and WLB are detected, one would know that the sample includes both target nucleic acid sequence A and target nucleic acid sequence B. In some embodiments, detection can occur through any of a variety of mobility dependent analytical techniques based on differential rates of migration between different analyte species. Exemplary mobility-dependent analysis techniques include electrophoresis, chromatography, mass spectroscopy, sedimentation, e.g., gradient centrifugation, field-flow fractionation, multi-stage extraction techniques, and the like. In some embodiments, mobility probes can be hybridized to amplification products, and the identity of the target polynucleotide determined via a mobility dependent analysis technique of the eluted mobility probes, as described for example in Published P.C.T. Application WO04/46344 to Rosenblum et al., and WO01/92579 to Wenz et al. In some embodiments, detection can be achieved by various microarrays and related software such as the Applied Biosystems Array System with the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer and other commercially available array systems available from Affymetrix, Agilent, Illumina, and Amersham Biosciences, among others (see also Gerry et al., J. Mol. Biol. 292:251-62, 1999; De Bellis et al., Minerva Biotec 14:247-52, 2002; and Stears et al., Nat. Med. 9:140-45, including supplements, 2003). It will also be appreciated that detection can comprise reporter groups that are incorporated into the reaction products, either as part of labeled primers or due to the incorporation of labeled dNTPs during an amplification, or attached to reaction products, for example but not limited to, via hybridization tag complements comprising reporter groups or via linker arms that are integral or attached to reaction products. Detection of unlabeled reaction products, for example using mass spectrometry, is also within the scope of the current teachings.
EXEMPLARY EMBODIMENTS
FIGS. 1A-1C depict certain compositions according to some embodiments of the present teachings. FIG. 1A, a miRNA molecule (1, dashed line) is depicted. FIG. 1B, a linker probe (2) is depicted, illustrating a 3′ target specific portion (3), a stem (4), and a loop (5). FIG. 1C, a miRNA hybridized to a linker probe is depicted, illustrating the 3′ target specific portion of the linker probe (3) hybridized to the 3′ end region of the miRNA (6).
As shown in FIGS. 2A-2D, a target polynucleotide (9, dotted line) is illustrated to show the relationship with various components of the linker probe (10), the detector probe (7), and the reverse primer (8), according to various non-limiting embodiments of the present teachings. For example as shown in FIG. 2A, in some embodiments the detector probe (7) can correspond with the 3′ end region of the target polynucleotide in the amplification product as well as a region upstream from the 3′ end region of the target polynucleotide in the amplification product. (Here, the detector probe is depicted as rectangle (7) with an F and a Q, symbolizing a TaqMan probe with a florophore (F) and a quencher (Q)). Also shown in FIG. 2A, the loop can correspond to the reverse primer (8). In some embodiments as shown in FIG. 2B, the detector probe (7) can correspond with a region of the amplification product corresponding with the 3′ end region of the target polynucleotide in the amplification product, as well as a region upstream from the 3′ end region of the target polynucleotide in the amplification product, as well as the linker probe stem in the amplification product. Also shown in FIG. 2B, the upstream region of the stem, as well as the loop, can correspond to the reverse primer (8). In some embodiments as shown in FIG. 2C, the detector probe can correspond to the amplification product in a manner similar to that shown in FIG. 2B, but the loop can correspond to the reverse primer (8). In some embodiments as shown in FIG. 2D, the detector probe (7) can correspond with the linker probe stem in the amplification product. Also shown in FIG. 2D, the upstream region of the stem, as well as the loop can correspond to the reverse primer (8). It will be appreciated that various related strategies for implementing the different functional regions of these compositions are possible in light of the present teachings, and that such derivations are routine to one having ordinary skill in the art without undue experimentation.
FIG. 3 depicts the nucleotide relationship for the micro RNA MiR-16 (boxed, 11) according to some embodiments of the present teachings. Shown here is the interrelationship of MiR-16 to a forward primer (12) (SEQ ID No. 781), a linker probe (13), a TaqMan detector probe (14) (SEQ ID No. 782), and a reverse primer (boxed, 15) (SEQ ID No. 783). The TaqMan probe comprises a 3′ minor groove binder (MGB), and a 5′ FAM florophore. It will be appreciated that in some embodiments of the present teachings the detector probes, such as for example TaqMan probes, can hybridize to either strand of an amplification product. For example, in some embodiments the detector probe can hybridize to the strand of the amplification product corresponding to the first strand synthesized. In some embodiments, the detector probe can hybridize to the strand of the amplification product corresponding to the second strand synthesized.
FIG. 4 depicts a single-plex assay design according to some embodiments of the present teachings. Here, a miRNA molecule (16) and a linker probe (17) are hybridized together (18). The 3′ end of the linker probe of the target-linker probe composition is extended to form an extension product (19) that can be amplified in a PCR. The PCR can comprise a miRNA specific forward primer (20) and a reverse primer (21). The detection of a detector probe (22) during the amplification allows for quantitation of the miRNA.
FIG. 5 depicts an overview of a multiplex assay design according to some embodiments of the present teachings. Here, a multiplexed hybridization and extension reaction is performed in a first reaction vessel (23). Thereafter, aliquots of the extension reaction products from the first reaction vessel are transferred into a plurality of amplification reactions (here, depicted as PCRs 1, 2, and 3) in a plurality of second reaction vessels. Each PCR can comprise a distinct primer pair and a distinct detector probe. In some embodiments, a distinct primer pair but the same detector probe can be present in each of a plurality of PCRs.
FIG. 6 depicts a multiplex assay design according to some embodiments of the present teachings. Here, three different miRNAs (24, 25, and 26) are queried in a hybridization reaction comprising three different linker probes (27, 28, and 29). Following hybridization and extension to form extension products (30, 31, and 32), the extension products are divided into three separate amplification reactions. (Though not explicitly shown, it will be appreciated that a number of copies of the molecules depicted by 30, 31, and 32 can be present, such that each of the three amplification reactions can have copies of 30, 31, and 32.) PCR 1 comprises a forward primer specific for miRNA 24 (33), PCR 2 comprises a forward primer specific for miRNA 25 (34), and PCR 3 comprises a forward primer specific for miRNA 26 (35). Each of the forward primers further comprise a non-complementary tail portion. PCR 1, PCR 2, and PCR 3 all comprise the same universal reverse primer 36. Further, PCR 1 comprises a distinct detector probe (37) that corresponds to the 3′ end region of miRNA 24 and the stem of linker probe 27, PCR 2 comprises a distinct detector probe (38) that corresponds to the 3′ end region of miRNA 25 and the stem of linker probe 28, and PCR 3 comprises a distinct detector probe (39) that corresponds to the 3′ region of miRNA 26 and the stem of linker probe 29.
The present teachings also contemplate reactions comprising configurations other than a linker probe. For example, in some embodiments, two hybridized molecules with a sticky end can be employed, wherein for example an overlapping 3′ sticky end hybridizes with the 3′ end region of the target polynucleotide. Some descriptions of two molecule configurations that can be employed in the present teachings can be found in Chen et al., U.S. Provisional Application 60/517,470. Viewed in light of the present teachings herein, one of skill in the art will appreciate that the approaches of Chen et al., can also be employed to result in extension reaction products that are longer that the target polynucleotide. These longer products can be detected with detector probes by, for example, taking advantage of the additional nucleotides introduced into the reaction products.
The present teachings also contemplate embodiments wherein the linker probe is ligated to the target polynucleotide, as described for example in Chen et al., U.S. Provisional Application 60/575,661, and the corresponding co-filed U.S. Provisional application co-filed herewith
Further, it will be appreciated that in some embodiments of the present teachings, the two molecule configurations in Chen et al., U.S. Provisional Application 60/517,470 can be applied in embodiments comprising the linker approaches discussed in Chen et al., U.S. Provisional Application 60/575,661.
Generally however, the loop structure of the present teachings will enhance the Tm of the target polynucleotide-linker probe duplex. Without being limited to any particular theory, this enhanced Tm could possibly be due to base stacking effects. Also, the characteristics of the looped linker probe of the present teachings can minimize nonspecific priming during the extension reaction, and/or a subsequent amplification reaction such as PCR. Further, the looped linker probe of the present teachings can better differentiate mature and precursor forms of miRNA, as illustrated infra in Example 6.
The present teachings also contemplate encoding and decoding reaction schemes, wherein a first encoding extension reaction is followed by a second decoding amplification reaction, as described for example in Livak et al., U.S. Provisional Application 60/556,162, Chen et al., U.S. Provisional Application 60/556,157, Andersen et al., U.S. Provisional Application 60/556,224, and Lao et al., U.S. Provisional Application 60/556,163.
The present teachings also contemplate a variety of strategies to minimize the number of different molecules in multiplexed amplification strategies, as described for example in Whitcombe et al., U.S. Pat. No. 6,270,967.
In certain embodiments, the present teachings also provide kits designed to expedite performing certain methods. In some embodiments, kits serve to expedite the performance of the methods of interest by assembling two or more components used in carrying out the methods. In some embodiments, kits may contain components in pre-measured unit amounts to minimize the need for measurements by end-users. In some embodiments, kits may include instructions for performing one or more methods of the present teachings. In certain embodiments, the kit components are optimized to operate in conjunction with one another.
For example, the present teachings provide a kit comprising, a reverse transcriptase and a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target-specific portion, wherein the 3′ target-specific portion corresponds to a miRNA. In some embodiments, the kits can comprise a DNA polymerase. In some embodiments, the kits can comprise a primer pair. In some embodiments, the kits can further comprise a forward primer specific for a miRNA, and, a universal reverse primer, wherein the universal reverse primer comprises a nucleotide of the loop of the linker probe. In some embodiments, the kits can comprise a plurality of primer pairs, wherein each primer pair is in one reaction vessel of a plurality of reaction vessels. In some embodiments, the kits can comprise a detector probe. In some embodiments, the detector probe comprises a nucleotide of the linker probe stem in the amplification product or a nucleotide of the linker probe stem complement in the amplification product, and the detector probe further comprises a nucleotide of the 3′ end region of the miRNA in the amplification product or a nucleotide of the 3′ end region of the miRNA complement in the amplification product.
The present teachings further contemplate kits comprising a means for hybridizing, a means for extending, a means for amplifying, a means for detecting, or combinations thereof.
While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings. Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the teachings in any way.
EXAMPLE 1
A single-plex reaction was performed in replicate for a collection of mouse miRNAs, and the effect of the presence or absence of ligase, as well as the presence or absence of reverse transcriptase, determined. The results are shown in Table 1 as Ct values.
First, a 6 ul reaction was set up comprising: 1 ul Reverse Transcription Enzyme Mix (Applied Biosystems part number 4340444) (or 1 ul dH2O), 0.5 ul T4 DNA Ligase (400 units/ul, NEB) (or 0.5 ul dH2O), 0.25 ul 2M KCl, 0.05 ul dNTPs (25 mM each), 0.25 ul T4 Kinase (10 units/ul, NEB), 1 ul 10×T4 DNA ligase buffer (NEB), 0.25 ul Applied Biosystems RNase Inhibitor (10 units/up, and 2.2 ul dH2O Next, 2 ul of the linker probe (0.25 uM) and RNA samples (2 ul of 0.25 ug/ul mouse lung total RNA (Ambion, product number 7818) were added. Next, the reaction was mixed, spun briefly, and placed on ice for 5 minutes.
The reaction was then incubated at 16 C for 30 minutes, 42 C for 30 minutes, followed by 85 C for 5 minutes, and then held at 4 C. The reactions were diluted 4 times by adding 30 ul of dH2O prior to the PCR amplification.
A 10 ul PCR amplification was then set up comprising: 2 ul of diluted reverse transcription reaction product, 1.3 ul 10 uM miRNA specific Forward Primer, 0.7 ul 10 uM Universal Reverse Primer, 0.2 ul TaqMan detector probe, 0.2 ul dNTPs (25 mM each), 0.6 ul dH2O, 5 ul 2× TaqMan master mix (Applied Biosystems, without UNG). The reaction was started with a 95 C step for 10 minutes. Then, 40 cycles were performed, each cycle comprising 95 C for 15 seconds, and 60 C for 1 minute. Table 1 indicates the results of this experiment.

















TABLE 1







Reverse







miRNA


Replicate
Ligase
transcriptase
Let-7a1
mir16
mir20
mir21
mir26a
mir30a
mir224
average


























Yes
Yes
16.8
16.0
19.1
16.8
15.0
21.3
27.3
18.9



Yes
No
38.7
31.3
39.9
31.9
30.1
33.3
40.0
35.0


I
No
Yes
18.0
14.6
18.3
16.2
14.0
21.3
26.4
18.4



No
No
40.0
36.6
40.0
40.0
33.8
39.2
40.0
38.5



Yes
Yes
17.1
16.2
19.3
17.0
15.1
21.4
27.3
19.1



Yes
No
38.9
31.2
37.6
32.1
30.4
33.4
39.4
34.7


II
No
Yes
18.4
14.8
18.7
16.6
14.3
21.5
26.7
18.7



No
No
40.0
36.1
40.0
40.0
34.1
40.0
40.0
38.6


Replicate
Yes
Yes
16.9
16.1
19.2
16.9
15.0
21.4
27.3
19.0


Average
Yes
No
38.8
31.2
38.8
32.0
30.3
33.4
39.7
34.9



No
Yes
18.2
14.7
18.5
16.4
14.1
21.4
26.6
18.6



No
No
40.0
36.4
40.0
40.0
34.0
39.6
40.0
40.0









Sequences of corresponding forward primers, reverse primer, and TaqMan probes are shown in Table 2.









TABLE 2








SEQ ID



miRNA ID
NO:
miRNA sequences





miR-16
1
uagcagcacguaaauauuggcg


miR-20
2
uaaagugcuuauagugcaggua


miR-21
3
uagcuuaucagacugauguuga


miR-22
4
aagcugccaguugaagaacugu


miR-26a
5
uucaaguaauccaggauaggcu


miR-29
6
cuagcaccaucugaaaucgguu


miR-30a
7
cuuucagucggauguuugcagc


miR-34
8
uggcagugucuuagcugguugu


miR-200b
9
cucuaauacugccugguaaugaug


miR-323
10
gcacauuacacggucgaccucu


miR-324-5
11
cgcauccccuagggcauuggugu


let-7a1
12
ugagguaguagguuguauaguu






SEQ ID



Linker probe
NO:
Linker probe sequences





miR-16linR6
13
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCGCCAA


miR20LinR6
14
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTACCTG


miR-21linR6
15
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACA


miR-22linR6
16
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAGTT


miR-26alinR6
17
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGCCTA


miR-29linR6
18
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACCGA


miR30LinR6
19
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTGCA


miR-34linR6
20
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAACC


miR-200blinR61
21
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCATCAT


miR-3231inR6
22
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGAGGT


miR-324-5linR6
23
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACACCA


let7aLinR6
24
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTAT






SEQ ID



Forward primer ID
NO:
Forward primer sequences





miR-16F55
25
CGCGCTAGCAGCACGTAAAT


miR-20F56
26
GCCGCTAAAGTGCTTATAGTGC


miR-21F56
27
GCCCGCTAGCTTATCAGACTGATG


miR-22F56
28
GCCTGAAGCTGCCAGTTGA


miR-26aF54
29
CCGGCGTTCAAGTAATCCAGGA


miR-29F56
30
GCCGCTAGCACCATCTGAAA


miR-30aF58
31
GCCCCTTTCAGTCGGATGTTT


miR-34F56
32
GCCCGTGGCAGTGTCTTAG


miR-200bF56
33
GCCCCTCTAATACTGCCTGG


miR-323F58
34
GCCACGCACATTACACGGTC


miR-324-5F56
35
GCCACCATCCCCTAGGGC


let-7a1F56
36
GCCGCTGAGGTAGTAGGTTGT






SEQ ID



TaqMan probe ID
NO:
TaqMan probe sequences





miR-16_Tq8F67
37
(6FAM)ATACGACCGCCAATAT(MGB)


miR20_Tq8F68
38
(6FAM)CTGGATACGACTACCTG(MGB)


miR-21_Tq8F68
39
(6FAM)CTGGATACGACTCAACA(MGB)


miR-22_Tq8F68
40
(6FAM)TGGATACGACACAGTTCT(MGB)


miR-26a_Tq8F69
41
(6FAM)TGGATACGACAGCCTATC(MGB)


miR-29_Tq8F68
42
(6FAM)TGGATACGACAACCGAT(MGB)


miR30_Tq8F68
43
(6FAM)CTGGATACGACGCTGC(MGB)


miR-34_Tq8F68
44
(6FAM)ATACGACACAACCAGC(MGB)


miR-200b_Tq8F67
45
(6FAM)ATACGACCATCATTACC(MGB)


miR-323_Tq8F67
46
(6FAM)CTGGATACGACAGAGGT(MGB)


miR-324-5Tq8F68
47
(6FAM)ATACGACACACCAATGC(MGB)


let7a_Tq8F68
48
(6FAM)TGGATACGACAACTATAC(MGB)






SEQ ID



Universal reverse primer ID
NO:
Reverse primer sequence





miR-UP-R67.8
49
GTGCAGGGTCCGAGGT









EXAMPLE 2
A multiplex (12-plex) assay was performed and the results compared to a corresponding collection of single-plex reactions. Additionally, the effect of the presence or absence of ligase, as well as the presence or absence of reverse transcriptase, was determined. The experiments were performed essentially the same as in Example 1, and the concentration of each linker in the 12-plex reaction was 0.05 uM, thereby resulting in a total linker probe concentration of 0.6 uM. Further, the diluted 12-plex reverse transcription product was split into 12 different PCR amplification reactions, wherein a miRNA forward primer and a universal reverse primer and a detector probe where in each amplification reaction. The miRNA sequences, Forward primers, and TaqMan detector probes are included in Table 2. The results are shown in Table 3.







TABLE 3







Singleplex vs. Multiplex Assay With Or Without T4 DNA Ligase












1-plex Ct
12-plex Ct
Ligation +















Ligation +
RT
Ligation +
RT
RT vs
1- vs. 12-


miRNA
RT
only
RT
only
RT only
plex
















let-7a1
17.8
16.3
17.6
17.0
1.0
−0.3


mir-16
16.0
15.1
16.1
15.3
0.9
−0.1


mir-20
19.3
18.7
19.8
19.5
0.4
−0.6


mir-21
17.0
15.8
17.1
16.3
1.0
−0.3


mir-22
21.6
20.4
21.4
20.7
1.0
−0.1


mir-26a
15.2
14.3
15.6
14.9
0.8
−0.4


mir-29
17.9
16.8
17.7
17.0
0.9
0.0


mir-30a
20.7
19.9
21.2
20.7
0.7
−0.7


mir-34
21.3
20.4
22.0
21.0
0.9
−0.6


mir-200b
19.9
19.2
21.1
20.2
0.8
−1.0


mir-323
32.5
31.2
33.6
32.3
1.3
−1.1


mir-324-5
24.7
23.1
25.0
24.4
1.1
−0.8


Average
20.3
19.3
20.7
19.9
0.9
−0.5









EXAMPLE 3
An experiment was performed to determine the effect of buffer conditions on reaction performance. In one set of experiments, a commercially available reverse transcription buffer from Applied Biosystems (part number 43400550) was employed in the hybridization and extension reaction. In a corresponding set of experiments, a commercially available T4 DNA ligase buffer (NEB) was employed in the hybridization and extension reaction. The experiments were performed as single-plex format essentially as described for Example 1, and each miRNA was done in triplicate. The results are shown in Table 4, comparing RT buffer (AB part #4340550) vs T4 DNA ligase buffer.











TABLE 4









RT Buffer
T4 DNA Ligase Buffer
RT vs

















I
II
III
Mean
I
II
III
Mean
T4 Buffer




















let-7a1
22.7
22.8
22.8
22.8
20.8
20.7
20.6
20.7
2.1


mir-16
18.4
18.5
18.6
18.5
17.7
17.8
17.9
17.8
0.7


mir-20
23.6
23.7
23.8
23.7
23.1
23.1
23.0
23.1
0.6


mir-21
20.4
20.4
20.5
20.4
19.4
19.3
19.2
19.3
1.1


mir-22
24.0
23.9
24.1
24.0
22.7
22.7
22.7
22.7
1.3


mir-26a
19.8
19.9
20.1
19.9
18.9
19.0
19.0
18.9
1.0


mir-29
21.3
21.3
21.4
21.3
20.5
20.6
20.5
20.5
0.8


mir-30a
24.4
24.4
24.4
24.4
23.6
23.4
23.6
23.5
0.9


mir-34
24.9
24.8
25.1
25.0
23.0
23.1
23.2
23.1
1.9


mir-200b
25.8
25.8
25.9
25.9
24.6
24.6
24.8
24.7
1.2


mir-323
34.6
34.5
34.8
34.6
34.7
34.2
34.5
34.5
0.2


mir-324-5
26.0
26.0
26.1
26.0
25.4
25.7
25.6
25.6
0.5


Average
23.8
23.8
24.0
23.9
22.9
22.8
22.9
22.9
1.0









EXAMPLE 4
An experiment was performed to examine the effect of ligase and kinase in a real-time miRNA amplification reaction. Here, twelve single-plex reactions were performed in duplicate, essentially as described in Example 1. Results are shown in Table 5.











TABLE 5









Ligase & Kinase

No Ligase/No Kinase














I
II
Mean
I
II
Mean



















let-7a1
17.7
17.9
17.8
16.2
16.4
16.3



mir-16
15.9
16.2
16.0
15.0
15.2
15.1



mir-20
19.1
19.6
19.3
18.6
18.9
18.7



mir-21
16.9
17.2
17.0
15.7
15.9
15.8



mir-22
21.4
21.7
21.6
20.3
20.5
20.4



mir-26a
15.0
15.4
15.2
14.3
14.4
14.3



mir-29
17.9
18.0
17.9
16.7
16.8
16.8



mir-30a
20.6
20.8
20.7
19.8
20.0
19.9



mir-34
21.1
21.5
21.3
20.4
20.5
20.4



mir-200b
19.8
20.0
19.9
19.2
19.3
19.2



mir-323
32.3
32.6
32.5
31.1
31.2
31.2



mir-324-5
24.6
24.8
24.7
23.0
23.3
23.1



Average
20.2
20.5
20.3
19.2
19.4
19.3










EXAMPLE 5
An experiment was performed to determine the effect of sample material on Ct values in a real-time miRNA amplification reaction. Here, cells, GuHCl lysate, Tris lysate, and Purified RNA were compared. The cells were NIH3T3 cells. The Purified RNA was collected using the commercially available mirVana mRNA isolation kit for Ambion (catalog number 1560). A Tris lysate, and a Guanidine lysate (GuHCl) (commercially available from Applied Biosystems), were prepared as follows:
For the Tris lysate, a 1× lysis buffer comprised 10 mM Tris-HCl, pH 8.0, 0.02% Sodium Azide, and 0.03% Tween-20. Trypsinized cells were pelleted by centrifugation at 1500 rpm for 5 minutes. The growth media was removed by aspiration, being careful that the cell pellet was not disturbed. PBS was added to bring the cells to 2×103 cells/ul. Next 10 ul of cell suspension was mixed with 10 ul of a 2× lysis buffer and spun briefly. The tubes were then immediately incubated for 5 minutes at 95 C, and then immediately placed in a chilled block on ice for 2 minutes. The tubes were then mixed well and spun briefly at full speed before use (or optionally, stored at −20 C).
For the GuHCl lysate, a 1× lysis buffer comprised 2.5M GuHCl, 150 mM MES pH 6.0, 200 mM NaCl, 0.75% Tween-20. Trypsinized cells were pelleted by centrifugation at 1500 rpm for 5 minutes. The growth media was removed by aspiration, being careful that the cell pellet was not disturbed. The cell pellet was then re-suspended in 1×PBS, Ca++ and Mg++ free to bring cells to 2×104 cells/uL. Then, 1 volume of 2× lysis buffer was added. To ensure complete nucleic acid release, this was followed by pipetting up and down ten times, followed by a brief spin. Results are shown in Table 6.
Similar results were obtained for a variety of cell lines, including NIH/3T3, OP9, A549, and HepG2 cells.









TABLE 6









Ct











miRNA ID
Cells
GuHCl lysate
Tris lysate
Purified RNA














let-7a1
24.9
31.3
28.2
31.5


mir-16
22.3
25.2
22.3
24.9


mir-20
22.7
26.0
24.1
26.1


mir-21
21.3
24.2
22.0
24.7


mir-22
30.3
28.6
27.2
28.8


mir-26a
25.6
31.0
27.9
31.4


mir-29
27.2
27.9
26.5
27.4


mir-30a
26.1
32.2
28.9
30.7


mir-34
26.8
30.3
26.4
27.4


mir-200b
40.0
40.0
40.0
40.0


mir-323
30.1
34.7
31.1
31.8


mir-324-5
28.6
29.7
28.3
29.3


Average
27.2
30.1
27.8
29.5









EXAMPLE 6
An experiment was performed to demonstrate the ability of the reaction to selectively quantity mature miRNA in the presence of precursor miRNA. Here, let-7a miRNA and mir-26b miRNA were queried in both mature form as well as in their precursor form. Experiments were performed essentially as described for Example 1 in the no ligase condition, done in triplicate, with varying amounts of target material as indicated. Results are shown in Table 7. The sequences examined were as follows:







Mature let-7a,


Seq ID NO: 50


UGAGGUAGUAGGUUGUAUAGUU


 


Precursor let-7a, (Note that the underlined


sequences corresponds to the Mature let-7a.)


SEQ ID NO: 51


GGGUGAGGUAGUAGGUUGUAUAGUUUGGGGCUCUGCCCUGCUAUGGGA


 


UAACUAUACAAUCUACUGUCUUUCCU


 


Mature mir-26b,


SEQ ID NO: 52


UUCAAGUAAUUCAGGAUAGGU


 


Precursor mir-26b of (Note that the underlined


sequences corresponds to the Mature mir-26b.)


SEQ ID NO: 53


CCGGGACCCAGUUCAAGUAAUUCAGGAUAGGUUGUGUGCUGUCCAGCCU


 


GUUCUCCAUUACUUGGCUCGGGGACCGG


















TABLE 7









Mouse
Synthetic
Synthetic




lung
miRNA
precursor
Assay specific for (CT)












Target
RNA (ng)
(fM)
(fM)
miRNA
Precursor















Let-7a
0
0
0
40.0 ± 0.0
40.0 ± 0.0


(let-7a3)
0
10
0
24.2 ± 0.3
40.0 ± 0.0



0
100
0
21.0 ± 0.2
40.0 ± 0.0



0
0
10
35.0 ± 1.0
25.0 ± 0.1



0
0
100
31.0 ± 0.1
21.5 ± 0.1



10
0
0
19.1 ± 0.4
40.0 ± 0.0


Mir-26b
0
0
0
40.0 ± 0.0
40.0 ± 0.0



0
10
0
23.1 ± 0.1
40.0 ± 0.0



0
100
0
19.7 ± 0.1
40.0 ± 0.0



0
0
10
32.9 ± 0.4
25.7 ± 0.0



0
0
100
28.9 ± 0.2
22.3 ± 0.0



10
0
0
20.5 ± 0.1
28.0 ± 0.2









EXAMPLE 7
An experiment was performed on synthetic let-7a miRNA to assess the number of 3′ nucleotides in the 3′ target specific portion of the linker probe that correspond with the 3′ end region of the miRNA. The experiment was performed as essentially as described supra for Example 1 for the no ligase condition, and results are shown in Table 8 as means and standard deviations of Ct values.







TABLE 8







miRNA assay components: let-7a


miRNA synthetic target: let-7a








No. 3′ ssDNA linker
CT values & statistics












probe target specific portion bases
I
II
III
Average
SD





7
29.4
29.1
29.3
29.3
0.1


6
30.1
29.9
30.2
30.1
0.2


5
33.9
33.2
33.8
33.6
0.4


4
40.0
39.2
40.0
39.7
0.4









In some embodiments, 3′ target specific portions of linker probes preferably comprise 5 nucleotides that correspond to the 3′ end region of miRNAs. For example, miR-26a and miR-26b differ by only 2 bases, one of which is the 3′ end nucleotide of miR-26a. Linker probes comprising 5 nucleotides at their 3′ target specific portions can be employed to selectively detect miR-26a versus miR-26b.

Additional strategies for using the linker probes of the present teachings in the context of single step assays, as well as in the context of short primer compositions, can be found in filed U.S. Provisional Application “Compositions, Methods, and Kits for Identifying and Quantitating Small RNA Molecules” by Lao and Straus, as well as in Elfaitouri et al., J. Clin. Virol. 2004, 30(2): 150-156.
The present teachings further contemplate linker probe compositions comprising 3′ target specific portions corresponding to any micro RNA sequence, including but without limitation, those sequences shown in Table 9, including C. elegans (cel), mouse (mmu), human (hsa), drosophila (dme), rat (rno), and rice (osa).








TABLE 9






SEQ ID



NO:
















cel-let-7
54


ugagguaguagguuguauaguu



 


cel-lin-4
55


ucccugagaccucaaguguga



 


cel-miR-1
56


uggaauguaaagaaguaugua



 


cel-miR-2
57


uaucacagccagcuuugaugugc



 


cel-miR-34
58


aggcagugugguuagcugguug



 


cel-miR-35
59


ucaccggguggaaacuagcagu



 


cel-miR-36
60


ucaccgggugaaaauucgcaug



 


cel-miR-37
61


ucaccgggugaacacuugcagu



 


cel-miR-38
62


ucaccgggagaaaaacuggagu



 


cel-miR-39
63


ucaccggguguaaaucagcuug



 


cel-miR-40
64


ucaccggguguacaucagcuaa



 


cel-miR-41
65


ucaccgggugaaaaaucaccua



 


cel-miR-42
66


caccggguuaacaucuacag



 


cel-miR-43
67


uaucacaguuuacuugcugucgc



 


cel-miR-44
68


ugacuagagacacauucagcu



 


cel-miR-45
69


ugacuagagacacauucagcu



 


cel-miR-46
70


ugucauggagucgcucucuuca



 


cel-miR-47
71


ugucauggaggcgcucucuuca



 


cel-miR-48
72


ugagguaggcucaguagaugcga



 


cel-miR-49
73


aagcaccacgagaagcugcaga



 


cel-miR-50
74


ugauaugucugguauucuuggguu



 


cel-miR-51
75


uacccguagcuccuauccauguu



 


cel-miR-52
76


cacccguacauauguuuccgugcu



 


cel-miR-53
77


cacccguacauuuguuuccgugcu



 


cel-miR-54
78


uacccguaaucuucauaauccgag



 


cel-miR-55
79


uacccguauaaguuucugcugag



 


cel-miR-56*
80


uggcggauccauuuuggguugua



 


cel-miR-56
81


uacccguaauguuuccgcugag



 


cel-miR-57
82


uacccuguagaucgagcugugugu



 


cel-miR-58
83


ugagaucguucaguacggcaau



 


cel-miR-59
84


ucgaaucguuuaucaggaugaug



 


cel-miR-60
85


uauuaugcacauuuucuaguuca



 


cel-miR-61
86


ugacuagaaccguuacucaucuc



 


cel-miR-62
87


ugauauguaaucuagcuuacag



 


cel-miR-63
88


uaugacacugaagcgaguuggaaa



 


cel-miR-64
89


uaugacacugaagcguuaccgaa



 


cel-miR-65
90


uaugacacugaagcguaaccgaa



 


cel-miR-66
91


caugacacugauuagggauguga



 


cel-miR-67
92


ucacaaccuccuagaaagaguaga



 


cel-miR-68
93


ucgaagacucaaaaguguaga



 


cel-miR-69
94


ucgaaaauuaaaaaguguaga



 


cel-miR-70
95


uaauacgucguugguguuuccau



 


cel-miR-71
96


ugaaacacauggguaguga



 


cel-miR-72
97


aggcaagauguuggcauagc



 


cel-miR-73
98


uggcaagauguaggcaguucagu



 


cel-miR-74
99


uggcaagaaauggcagucuaca



 


cel-miR-75
100


uuaaagcuaccaaccggcuuca



 


cel-miR-76
101


uucguuguugaugaagccuuga



 


cel-miR-77
102


uucaucaggccauagcugucca



 


cel-miR-78
103


uggaggccugguuguuugugc



 


cel-miR-79
104


auaaagcuagguuaccaaagcu



 


cel-miR-227
105


agcuuucgacaugauucugaac



 


cel-miR-80
106


ugagaucauuaguugaaagccga



 


cel-miR-81
107


ugagaucaucgugaaagcuagu



 


cel-miR-82
108


ugagaucaucgugaaagccagu



 


cel-miR-83
109


uagcaccauauaaauucaguaa



 


cel-miR-84
110


ugagguaguauguaauauugua



 


cel-miR-85
111


uacaaaguauuugaaaagucgugc



 


cel-miR-86
112


uaagugaaugcuuugccacaguc



 


cel-miR-87
113


gugagcaaaguuucaggugu



 


cel-miR-90
114


ugauauguuguuugaaugcccc



 


cel-miR-124
115


uaaggcacgcggugaaugcca



 


cel-miR-228
116


aauggcacugcaugaauucacgg



 


cel-miR-229
117


aaugacacugguuaucuuuuccaucgu



 


cel-miR-230
118


guauuaguugugcgaccaggaga



 


cel-miR-231
119


uaagcucgugaucaacaggcagaa



 


cel-miR-232
120


uaaaugcaucuuaacugcgguga



 


cel-miR-233
121


uugagcaaugcgcaugugcggga



 


cel-miR-234
122


uuauugcucgagaauacccuu



 


cel-miR-235
123


uauugcacucuccccggccuga



 


cel-miR-236
124


uaauacugucagguaaugacgcu



 


cel-miR-237
125


ucccugagaauucucgaacagcuu



 


cel-miR-238
126


uuuguacuccgaugccauucaga



 


cel-miR-239a
127


uuuguacuacacauagguacugg



 


cel-miR-239b
128


uuguacuacacaaaaguacug



 


cel-miR-240
129


uacuggcccccaaaucuucgcu



 


cel-miR-241
130


ugagguaggugcgagaaauga



 


cel-miR-242
131


uugcguaggccuuugcuucga



 


cel-miR-243
132


cgguacgaucgcggcgggauauc



 


cel-miR-244
133


ucuuugguuguacaaagugguaug



 


cel-miR-245
134


auugguccccuccaaguagcuc



 


cel-miR-246
135


uuacauguuucggguaggagcu



 


cel-miR-247
136


ugacuagagccuauucucuucuu



 


cel-miR-248
137


uacacgugcacggauaacgcuca



 


cel-miR-249
138


ucacaggacuuuugagcguugc



 


cel-miR-250
139


ucacagucaacuguuggcaugg



 


cel-miR-251
140


uuaaguaguggugccgcucuuauu



 


cel-miR-252
141


uaaguaguagugccgcagguaac



 


cel-miR-253
142


cacaccucacuaacacugacc



 


cel-miR-254
143


ugcaaaucuuucgcgacuguagg



 


cel-miR-256
144


uggaaugcauagaagacugua



 


cel-miR-257
145


gaguaucaggaguacccaguga



 


cel-miR-258
146


gguuuugagaggaauccuuuu



 


cel-miR-259
147


aaaucucauccuaaucuggua



 


cel-miR-260
148


gugaugucgaacucuuguag



 


cel-miR-261
149


uagcuuuuuaguuuucacg



 


cel-miR-262
150


guuucucgauguuuucugau



 


cel-miR-264
151


ggcgggugguuguuguuaug



 


cel-miR-265
152


ugagggaggaagggugguau



 


cel-miR-266
153


aggcaagacuuuggcaaagc



 


cel-miR-267
154


cccgugaagugucugcugca



 


cel-miR-268
155


ggcaagaauuagaagcaguuuggu



 


cel-miR-269
156


ggcaagacucuggcaaaacu



 


cel-miR-270
157


ggcaugauguagcaguggag



 


cel-miR-271
158


ucgccgggugggaaagcauu



 


cel-miR-272
159


uguaggcauggguguuug



 


cel-miR-273
160


ugcccguacugugucggcug



 


cel-miR-353
161


caauugccauguguugguauu



 


cel-miR-354
162


accuuguuuguugcugcuccu



 


cel-miR-355
163


uuuguuuuagccugagcuaug



 


cel-miR-356
164


uugagcaacgcgaacaaauca



 


cel-miR-357
165


uaaaugccagucguugcagga



 


cel-miR-358
166


caauugguaucccugucaagg



 


cel-miR-359
167


ucacuggucuuucucugacga



 


cel-miR-360
168


ugaccguaaucccguucacaa



 


cel-lsy-6
169


uuuuguaugagacgcauuucg



 


cel-miR-392
170


uaucaucgaucacgugugauga



 


hsa-let-7a
171


ugagguaguagguuguauaguu



 


hsa-let-7b
172


ugagguaguagguugugugguu



 


hsa-let-7c
173


ugagguaguagguuguaugguu



 


hsa-let-7d
174


agagguaguagguugcauagu



 


hsa-let-7e
175


ugagguaggagguuguauagu



 


hsa-let-7f
176


ugagguaguagauuguauaguu



 


hsa-miR-15a
177


uagcagcacauaaugguuugug



 


hsa-miR-16
178


uagcagcacguaaauauuggcg



 


hsa-miR-17-5p
179


caaagugcuuacagugcagguagu



 


hsa-miR-17-3p
180


acugcagugaaggcacuugu



 


hsa-miR-18
181


uaaggugcaucuagugcagaua



 


hsa-miR-19a
182


ugugcaaaucuaugcaaaacuga



 


hsa-miR-19b
183


ugugcaaauccaugcaaaacuga



 


hsa-miR-20
184


uaaagugcuuauagugcaggua



 


hsa-miR-21
185


uagcuuaucagacugauguuga



 


hsa-miR-22
186


aagcugccaguugaagaacugu



 


hsa-miR-23a
187


aucacauugccagggauuucc



 


hsa-miR-189
188


gugccuacugagcugauaucagu



 


hsa-miR-24
189


uggcucaguucagcaggaacag



 


hsa-miR-25
190


cauugcacuugucucggucuga



 


hsa-miR-26a
191


uucaaguaauccaggauaggcu



 


hsa-miR-26b
192


uucaaguaauucaggauaggu



 


hsa-miR-27a
193


uucacaguggcuaaguuccgcc



 


hsa-miR-28
194


aaggagcucacagucuauugag



 


hsa-miR-29a
195


cuagcaccaucugaaaucgguu



 


hsa-miR-30a*
196


uguaaacauccucgacuggaagc



 


hsa-miR-30a
197


cuuucagucggauguuugcagc



 


hsa-miR-31
198


ggcaagaugcuggcauagcug



 


hsa-miR-32
199


uauugcacauuacuaaguugc



 


hsa-miR-33
200


gugcauuguaguugcauug



 


hsa-miR-92
201


uauugcacuugucccggccugu



 


hsa-miR-93
202


aaagugcuguucgugcagguag



 


hsa-miR-95
203


uucaacggguauuuauugagca



 


hsa-miR-96
204


uuuggcacuagcacauuuuugc



 


hsa-miR-98
205


ugagguaguaaguuguauuguu



 


hsa-miR-99a
206


aacccguagauccgaucuugug



 


hsa-miR-100
207


aacccguagauccgaacuugug



 


hsa-miR-101
208


uacaguacugugauaacugaag



 


hsa-miR-29b
209


uagcaccauuugaaaucagu



 


hsa-miR-103
210


agcagcauuguacagggcuauga



 


hsa-miR-105
211


ucaaaugcucagacuccugu



 


hsa-miR-106a
212


aaaagugcuuacagugcagguagc



 


hsa-miR-107
213


agcagcauuguacagggcuauca



 


hsa-miR-192
214


cugaccuaugaauugacagcc



 


hsa-miR-196
215


uagguaguuucauguuguugg



 


hsa-miR-197
216


uucaccaccuucuccacccagc



 


hsa-miR-198
217


gguccagaggggagauagg



 


hsa-miR-199a
218


cccaguguucagacuaccuguuc



 


hsa-miR-199a*
219


uacaguagucugcacauugguu



 


hsa-miR-208
220


auaagacgagcaaaaagcuugu



 


hsa-miR-148a
221


ucagugcacuacagaacuuugu



 


hsa-miR-30c
222


uguaaacauccuacacucucagc



 


hsa-miR-30d
223


uguaaacauccccgacuggaag



 


hsa-miR-139
224


ucuacagugcacgugucu



 


hsa-miR-147
225


guguguggaaaugcuucugc



 


hsa-miR-7
226


uggaagacuagugauuuuguu



 


hsa-miR-10a
227


uacccuguagauccgaauuugug



 


hsa-miR-10b
228


uacccuguagaaccgaauuugu



 


hsa-miR-34a
229


uggcagugucuuagcugguugu



 


hsa-miR-181a
230


aacauucaacgcugucggugagu



 


hsa-miR-181b
231


aacauucauugcugucgguggguu



 


hsa-miR-181c
232


aacauucaaccugucggugagu



 


hsa-miR-182
233


uuuggcaaugguagaacucaca



 


hsa-miR-182*
234


ugguucuagacuugccaacua



 


hsa-miR-183
235


uauggcacugguagaauucacug



 


hsa-miR-187
236


ucgugucuuguguugcagccg



 


hsa-miR-199b
237


cccaguguuuagacuaucuguuc



 


hsa-miR-203
238


gugaaauguuuaggaccacuag



 


hsa-miR-204
239


uucccuuugucauccuaugccu



 


hsa-miR-205
240


uccuucauuccaccggagucug



 


hsa-miR-210
241


cugugcgugugacagcggcug



 


hsa-miR-211
242


uucccuuugucauccuucgccu



 


hsa-miR-212
243


uaacagucuccagucacggcc



 


hsa-miR-213
244


accaucgaccguugauuguacc



 


hsa-miR-214
245


acagcaggcacagacaggcag



 


hsa-miR-215
246


augaccuaugaauugacagac



 


hsa-miR-216
247


uaaucucagcuggcaacugug



 


hsa-miR-217
248


uacugcaucaggaacugauuggau



 


hsa-miR-218
249


uugugcuugaucuaaccaugu



 


hsa-miR-219
250


ugauuguccaaacgcaauucu



 


hsa-miR-220
251


ccacaccguaucugacacuuu



 


hsa-miR-221
252


agcuacauugucugcuggguuuc



 


hsa-miR-222
253


agcuacaucuggcuacugggucuc



 


hsa-miR-223
254


ugucaguuugucaaauacccc



 


hsa-miR-224
255


caagucacuagugguuccguuua



 


hsa-miR-200b
256


cucuaauacugccugguaaugaug



 


hsa-let-7g
257


ugagguaguaguuuguacagu



 


hsa-let-7i
258


ugagguaguaguuugugcu



 


hsa-miR-1
259


uggaauguaaagaaguaugua



 


hsa-miR-15b
260


uagcagcacaucaugguuuaca



 


hsa-miR-23b
261


aucacauugccagggauuaccac



 


hsa-miR-27b
262


uucacaguggcuaaguucug



 


hsa-miR-30b
263


uguaaacauccuacacucagc



 


hsa-miR-122a
264


uggagugugacaaugguguuugu



 


hsa-miR-124a
265


uuaaggcacgcggugaaugcca



 


hsa-miR-125b
266


ucccugagacccuaacuuguga



 


hsa-miR-128a
267


ucacagugaaccggucucuuuu



 


hsa-miR-130a
268


cagugcaauguuaaaagggc



 


hsa-miR-132
269


uaacagucuacagccauggucg



 


hsa-miR-133a
270


uugguccccuucaaccagcugu



 


hsa-miR-135a
271


uauggcuuuuuauuccuauguga



 


hsa-miR-137
272


uauugcuuaagaauacgcguag



 


hsa-miR-138
273


agcugguguugugaauc



 


hsa-miR-140
274


agugguuuuacccuaugguag



 


hsa-miR-141
275


aacacugucugguaaagaugg



 


hsa-miR-142-5p
276


cauaaaguagaaagcacuac



 


hsa-miR-142-3p
277


uguaguguuuccuacuuuaugga



 


hsa-miR-143
278


ugagaugaagcacuguagcuca



 


hsa-miR-144
279


uacaguauagaugauguacuag



 


hsa-miR-145
280


guccaguuuucccaggaaucccuu



 


hsa-miR-152
281


ucagugcaugacagaacuugg



 


hsa-miR-153
282


uugcauagucacaaaaguga



 


hsa-miR-191
283


caacggaaucccaaaagcagcu



 


hsa-miR-9
284


ucuuugguuaucuagcuguauga



 


hsa-miR-9*
285


uaaagcuagauaaccgaaagu



 


hsa-miR-125a
286


ucccugagacccuuuaaccugug



 


hsa-miR-126*
287


cauuauuacuuuugguacgcg



 


hsa-miR-126
288


ucguaccgugaguaauaaugc



 


hsa-miR-127
289


ucggauccgucugagcuuggcu



 


hsa-miR-129
290


cuuuuugcggucugggcuugc



 


hsa-miR-134
291


ugugacugguugaccagaggg



 


hsa-miR-136
292


acuccauuuguuuugaugaugga



 


hsa-miR-146
293


ugagaacugaauuccauggguu



 


hsa-miR-149
294


ucuggcuccgugucuucacucc



 


hsa-miR-150
295


ucucccaacccuuguaccagug



 


hsa-miR-154
296


uagguuauccguguugccuucg



 


hsa-miR-184
297


uggacggagaacugauaagggu



 


hsa-miR-185
298


uggagagaaaggcaguuc



 


hsa-miR-186
299


caaagaauucuccuuuugggcuu



 


hsa-miR-188
300


caucccuugcaugguggagggu



 


hsa-miR-190
301


ugauauguuugauauauuaggu



 


hsa-miR-193
302


aacuggccuacaaagucccag



 


hsa-miR-194
303


uguaacagcaacuccaugugga



 


hsa-miR-195
304


uagcagcacagaaauauuggc



 


hsa-miR-206
305


uggaauguaaggaagugugugg



 


hsa-miR-320
306


aaaagcuggguugagagggcgaa



 


hsa-miR-321
307


uaagccagggauuguggguuc



 


hsa-miR-200c
308


aauacugccggguaaugaugga



 


hsa-miR-155
309


uuaaugcuaaucgugauagggg



 


hsa-miR-128b
310


ucacagugaaccggucucuuuc



 


hsa-miR-106b
311


uaaagugcugacagugcagau



 


hsa-miR-29c
312


uagcaccauuugaaaucgguua



 


hsa-miR-200a
313


uaacacugucugguaacgaugu



 


hsa-miR-302
314


uaagugcuuccauguuuugguga



 


hsa-miR-34b
315


aggcagugucauuagcugauug



 


hsa-miR-34c
316


aggcaguguaguuagcugauug



 


hsa-miR-299
317


ugguuuaccgucccacauacau



 


hsa-miR-301
318


cagugcaauaguauugucaaagc



 


hsa-miR-99b
319


cacccguagaaccgaccuugcg



 


hsa-miR-296
320


agggcccccccucaauccugu



 


hsa-miR-130b
321


cagugcaaugaugaaagggcau



 


hsa-miR-30e
322


uguaaacauccuugacugga



 


hsa-miR-340
323


uccgucucaguuacuuuauagcc



 


hsa-miR-330
324


gcaaagcacacggccugcagaga



 


hsa-miR-328
325


cuggcccucucugcccuuccgu



 


hsa-miR-342
326


ucucacacagaaaucgcacccguc



 


hsa-miR-337
327


uccagcuccuauaugaugccuuu



 


hsa-miR-323
328


gcacauuacacggucgaccucu



 


hsa-miR-326
329


ccucugggcccuuccuccag



 


hsa-miR-151
330


acuagacugaagcuccuugagg



 


hsa-miR-135b
331


uauggcuuuucauuccuaugug



 


hsa-miR-148b
332


ucagugcaucacagaacuuugu



 


hsa-miR-331
333


gccccugggccuauccuagaa



 


hsa-miR-324-5p
334


cgcauccccuagggcauuggugu



 


hsa-miR-324-3p
335


ccacugccccaggugcugcugg



 


hsa-miR-338
336


uccagcaucagugauuuuguuga



 


hsa-miR-339
337


ucccuguccuccaggagcuca



 


hsa-miR-335
338


ucaagagcaauaacgaaaaaugu



 


hsa-miR-133b
339


uugguccccuucaaccagcua



 


osa-miR156
340


ugacagaagagagugagcac



 


osa-miR160
341


ugccuggcucccuguaugcca



 


osa-miR162
342


ucgauaaaccucugcauccag



 


osa-miR164
343


uggagaagcagggcacgugca



 


osa-miR166
344


ucggaccaggcuucauucccc



 


osa-miR167
345


ugaagcugccagcaugaucua



 


osa-miR169
346


cagccaaggaugacuugccga



 


osa-miR171
347


ugauugagccgcgccaauauc



 


mmu-let-7g
348


ugagguaguaguuuguacagu



 


mmu-let-7i
349


ugagguaguaguuugugcu



 


mmu-miR-1
350


uggaauguaaagaaguaugua



 


mmu-miR-15b
351


uagcagcacaucaugguuuaca



 


mmu-miR-23b
352


aucacauugccagggauuaccac



 


mmu-miR-27b
353


uucacaguggcuaaguucug



 


mmu-miR-29b
354


uagcaccauuugaaaucagugu



 


mmu-miR-30a*
355


uguaaacauccucgacuggaagc



 


mmu-miR-30a
356


cuuucagucggauguuugcagc



 


mmu-miR-30b
357


uguaaacauccuacacucagc



 


mmu-miR-99a
358


acccguagauccgaucuugu



 


mmu-miR-99b
359


cacccguagaaccgaccuugcg



 


mmu-miR-101
360


uacaguacugugauaacuga



 


mmu-miR-124a
361


uuaaggcacgcggugaaugcca



 


mmu-miR-125a
362


ucccugagacccuuuaaccugug



 


mmu-miR-125b
363


ucccugagacccuaacuuguga



 


mmu-miR-126*
364


cauuauuacuuuugguacgcg



 


mmu-miR-126
365


ucguaccgugaguaauaaugc



 


mmu-miR-127
366


ucggauccgucugagcuuggcu



 


mmu-miR-128a
367


ucacagugaaccggucucuuuu



 


mmu-miR-130a
368


cagugcaauguuaaaagggc



 


mmu-miR-9
369


ucuuugguuaucuagcuguauga



 


mmu-miR-9*
370


uaaagcuagauaaccgaaagu



 


mmu-miR-132
371


uaacagucuacagccauggucg



 


mmu-miR-133a
372


uugguccccuucaaccagcugu



 


mmu-miR-134
373


ugugacugguugaccagaggg



 


mmu-miR-135a
374


uauggcuuuuuauuccuauguga



 


mmu-miR-136
375


acuccauuuguuuugaugaugga



 


mmu-miR-137
376


uauugcuuaagaauacgcguag



 


mmu-miR-138
377


agcugguguugugaauc



 


mmu-miR-140
378


agugguuuuacccuaugguag



 


mmu-miR-141
379


aacacugucugguaaagaugg



 


mmu-miR-142-5p
380


cauaaaguagaaagcacuac



 


mmu-miR-142-3p
381


uguaguguuuccuacuuuaugg



 


mmu-miR-144
382


uacaguauagaugauguacuag



 


mmu-miR-145
383


guccaguuuucccaggaaucccuu



 


mmu-miR-146
384


ugagaacugaauuccauggguu



 


mmu-miR-149
385


ucuggcuccgugucuucacucc



 


mmu-miR-150
386


ucucccaacccuuguaccagug



 


mmu-miR-151
387


cuagacugaggcuccuugagg



 


mmu-miR-152
388


ucagugcaugacagaacuugg



 


mmu-miR-153
389


uugcauagucacaaaaguga



 


mmu-miR-154
390


uagguuauccguguugccuucg



 


mmu-miR-155
391


uuaaugcuaauugugauagggg



 


mmu-miR-10b
392


cccuguagaaccgaauuugugu



 


mmu-miR-129
393


cuuuuugcggucugggcuugcu



 


mmu-miR-181a
394


aacauucaacgcugucggugagu



 


mmu-miR-182
395


uuuggcaaugguagaacucaca



 


mmu-miR-183
396


uauggcacugguagaauucacug



 


mmu-miR-184
397


uggacggagaacugauaagggu



 


mmu-miR-185
398


uggagagaaaggcaguuc



 


mmu-miR-186
399


caaagaauucuccuuuugggcuu



 


mmu-miR-187
400


ucgugucuuguguugcagccgg



 








mmu-miR-188
401


caucccuugcaugguggagggu



 


mmu-miR-189
402


gugccuacugagcugauaucagu



 


mmu-miR-24
403


uggcucaguucagcaggaacag



 


mmu-miR-190
404


ugauauguuugauauauuaggu



 


mmu-miR-191
405


caacggaaucccaaaagcagcu



 


mmu-miR-193
406


aacuggccuacaaagucccag



 


mmu-miR-194
407


uguaacagcaacuccaugugga



 


mmu-miR-195
408


uagcagcacagaaauauuggc



 


mmu-miR-199a
409


cccaguguucagacuaccuguuc



 


mmu-miR-199a*
410


uacaguagucugcacauugguu



 


mmu-miR-200b
411


uaauacugccugguaaugaugac



 


mmu-miR-201
412


uacucaguaaggcauuguucu



 


mmu-miR-202
413


agagguauagcgcaugggaaga



 


mmu-miR-203
414


ugaaauguuuaggaccacuag



 


mmu-miR-204
415


uucccuuugucauccuaugccug



 


mmu-miR-205
416


uccuucauuccaccggagucug



 


mmu-miR-206
417


uggaauguaaggaagugugugg



 


mmu-miR-207
418


gcuucuccuggcucuccucccuc



 


mmu-miR-122a
419


uggagugugacaaugguguuugu



 


mmu-miR-143
420


ugagaugaagcacuguagcuca



 


mmu-miR-30e
421


uguaaacauccuugacugga



 


mmu-miR-290
422


cucaaacuaugggggcacuuuuu



 


mmu-miR-291-5p
423


caucaaaguggaggcccucucu



 


mmu-miR-291-3p
424


aaagugcuuccacuuugugugcc



 


mmu-miR-292-5p
425


acucaaacugggggcucuuuug



 


mmu-miR-292-3p
426


aagugccgccagguuuugagugu



 


mmu-miR-293
427


agugccgcagaguuuguagugu



 


mmu-miR-294
428


aaagugcuucccuuuugugugu



 


mmu-miR-295
429


aaagugcuacuacuuuugagucu



 


mmu-miR-296
430


agggcccccccucaauccugu



 


mmu-miR-297
431


auguaugugugcaugugcaug



 


mmu-miR-298
432


ggcagaggagggcuguucuucc



 


mmu-miR-299
433


ugguuuaccgucccacauacau



 


mmu-miR-300
434


uaugcaagggcaagcucucuuc



 


mmu-miR-301
435


cagugcaauaguauugucaaagc



 


mmu-miR-302
436


uaagugcuuccauguuuugguga



 


mmu-miR-34c
437


aggcaguguaguuagcugauugc



 


mmu-miR-34b
438


uaggcaguguaauuagcugauug



 


mmu-let-7d
439


agagguaguagguugcauagu



 


mmu-let-7d*
440


cuauacgaccugcugccuuucu



 


mmu-miR-106a
441


caaagugcuaacagugcaggua



 


mmu-miR-106b
442


uaaagugcugacagugcagau



 


mmu-miR-130b
443


cagugcaaugaugaaagggcau



 


mmu-miR-19b
444


ugugcaaauccaugcaaaacuga



 


mmu-miR-30c
445


uguaaacauccuacacucucagc



 


mmu-miR-30d
446


uguaaacauccccgacuggaag



 


mmu-miR-148a
447


ucagugcacuacagaacuuugu



 


mmu-miR-192
448


cugaccuaugaauugaca



 


mmu-miR-196
449


uagguaguuucauguuguugg



 


mmu-miR-200a
450


uaacacugucugguaacgaugu



 


mmu-miR-208
451


auaagacgagcaaaaagcuugu



 


mmu-let-7a
452


ugagguaguagguuguauaguu



 


mmu-let-7b
453


ugagguaguagguugugugguu



 


mmu-let-7c
454


ugagguaguagguuguaugguu



 


mmu-let-7e
455


ugagguaggagguuguauagu



 


mmu-let-7f
456


ugagguaguagauuguauaguu



 


mmu-miR-15a
457


uagcagcacauaaugguuugug



 


mmu-miR-16
458


uagcagcacguaaauauuggcg



 


mmu-miR-18
459


uaaggugcaucuagugcagaua



 


mmu-miR-20
460


uaaagugcuuauagugcagguag



 


mmu-miR-21
461


uagcuuaucagacugauguuga



 


mmu-miR-22
462


aagcugccaguugaagaacugu



 


mmu-miR-23a
463


aucacauugccagggauuucc



 


mmu-miR-26a
464


uucaaguaauccaggauaggcu



 


mmu-miR-26b
465


uucaaguaauucaggauagguu



 


mmu-miR-29a
466


cuagcaccaucugaaaucgguu



 


mmu-miR-29c
467


uagcaccauuugaaaucgguua



 


mmu-miR-27a
468


uucacaguggcuaaguuccgc



 


mmu-miR-31
469


aggcaagaugcuggcauagcug



 


mmu-miR-92
470


uauugcacuugucccggccug



 


mmu-miR-93
471


caaagugcuguucgugcagguag



 


mmu-miR-96
472


uuuggcacuagcacauuuuugcu



 


mmu-miR-34a
473


uggcagugucuuagcugguuguu



 


mmu-miR-98
474


ugagguaguaaguuguauuguu



 


mmu-miR-103
475


agcagcauuguacagggcuauga



 


mmu-miR-323
476


gcacauuacacggucgaccucu



 


mmu-miR-324-5p
477


cgcauccccuagggcauuggugu



 


mmu-miR-324-3p
478


ccacugccccaggugcugcugg



 


mmu-miR-325
479


ccuaguaggugcucaguaagugu



 


mmu-miR-326
480


ccucugggcccuuccuccagu



 


mmu-miR-328
481


cuggcccucucugcccuuccgu



 


mmu-miR-329
482


aacacacccagcuaaccuuuuu



 


mmu-miR-330
483


gcaaagcacagggccugcagaga



 


mmu-miR-331
484


gccccugggccuauccuagaa



 


mmu-miR-337
485


uucagcuccuauaugaugccuuu



 


mmu-miR-338
486


uccagcaucagugauuuuguuga



 


mmu-miR-339
487


ucccuguccuccaggagcuca



 


mmu-miR-340
488


uccgucucaguuacuuuauagcc



 


mmu-miR-341
489


ucgaucggucggucggucagu



 


mmu-miR-342
490


ucucacacagaaaucgcacccguc



 


mmu-miR-344
491


ugaucuagccaaagccugacugu



 


mmu-miR-345
492


ugcugaccccuaguccagugc



 


mmu-miR-346
493


ugucugcccgagugccugccucu



 


mmu-miR-350
494


uucacaaagcccauacacuuucac



 


mmu-miR-135b
495


uauggcuuuucauuccuaugug



 


mmu-miR-101b
496


uacaguacugugauagcugaag



 


mmu-miR-107
497


agcagcauuguacagggcuauca



 


mmu-miR-10a
498


uacccuguagauccgaauuugug



 


mmu-miR-17-5p
499


caaagugcuuacagugcagguagu



 


mmu-miR-17-3p
500


acugcagugagggcacuugu



 


mmu-miR-19a
501


ugugcaaaucuaugcaaaacuga



 


mmu-miR-25
502


cauugcacuugucucggucuga



 


mmu-miR-28
503


aaggagcucacagucuauugag



 


mmu-miR-32
504


uauugcacauuacuaaguugc



 


mmu-miR-100
505


aacccguagauccgaacuugug



 


mmu-miR-139
506


ucuacagugcacgugucu



 


mmu-miR-200c
507


aauacugccggguaaugaugga



 


mmu-miR-210
508


cugugcgugugacagcggcug



 


mmu-miR-212
509


uaacagucuccagucacggcc



 


mmu-miR-213
510


accaucgaccguugauuguacc



 


mmu-miR-214
511


acagcaggcacagacaggcag



 


mmu-miR-216
512


uaaucucagcuggcaacugug



 


mmu-miR-218
513


uugugcuugaucuaaccaugu



 


mmu-miR-219
514


ugauuguccaaacgcaauucu



 


mmu-miR-223
515


ugucaguuugucaaauacccc



 


mmu-miR-320
516


aaaagcuggguugagagggcgaa



 


mmu-miR-321
517


uaagccagggauuguggguuc



 


mmu-miR-33
518


gugcauuguaguugcauug



 


mmu-miR-211
519


uucccuuugucauccuuugccu



 


mmu-miR-221
520


agcuacauugucugcuggguuu



 


mmu-miR-222
521


agcuacaucuggcuacugggucu



 


mmu-miR-224
522


uaagucacuagugguuccguuua



 


mmu-miR-199b
523


cccaguguuuagacuaccuguuc



 


mmu-miR-181b
524


aacauucauugcugucgguggguu



 


mmu-miR-181c
525


aacauucaaccugucggugagu



 


mmu-miR-128b
526


ucacagugaaccggucucuuuc



 


mmu-miR-7
527


uggaagacuagugauuuuguu



 


mmu-miR-7b
528


uggaagacuugugauuuuguu



 


mmu-miR-217
529


uacugcaucaggaacugacuggau



 


mmu-miR-133b
530


uugguccccuucaaccagcua



 


mmu-miR-215
531


augaccuaugauuugacagac



 


dme-miR-1
532


uggaauguaaagaaguauggag



 


dme-miR-2a
533


uaucacagccagcuuugaugagc



 


dme-miR-2b
534


uaucacagccagcuuugaggagc



 


dme-miR-3
535


ucacugggcaaagugugucuca



 


dme-miR-4
536


auaaagcuagacaaccauuga



 


dme-miR-5
537


aaaggaacgaucguugugauaug



 


dme-miR-6
538


uaucacaguggcuguucuuuuu



 


dme-miR-7
539


uggaagacuagugauuuuguugu



 


dme-miR-8
540


uaauacugucagguaaagauguc



 


dme-miR-9a
541


ucuuugguuaucuagcuguauga



 


dme-miR-10
542


acccuguagauccgaauuugu



 


dme-miR-11
543


caucacagucugaguucuugc



 


dme-miR-12
544


ugaguauuacaucagguacuggu



 


dme-miR-13a
545


uaucacagccauuuugaugagu



 


dme-miR-13b
546


uaucacagccauuuugacgagu



 


dme-miR-14
547


ucagucuuuuucucucuccua



 


dme-miR-263a
548


guuaauggcacuggaagaauucac



 


dme-miR-184*
549


ccuuaucauucucucgccccg



 


dme-miR-184
550


uggacggagaacugauaagggc



 


dme-miR-274
551


uuuugugaccgacacuaacggguaau



 


dme-miR-275
552


ucagguaccugaaguagcgcgcg



 


dme-miR-92a
553


cauugcacuugucccggccuau



 


dme-miR-219
554


ugauuguccaaacgcaauucuug



 


dme-miR-276a*
555


cagcgagguauagaguuccuacg



 


dme-miR-276a
556


uaggaacuucauaccgugcucu



 


dme-miR-277
557


uaaaugcacuaucugguacgaca



 


dme-miR-278
558


ucggugggacuuucguccguuu



 


dme-miR-133
559


uugguccccuucaaccagcugu



 


dme-miR-279
560


ugacuagauccacacucauuaa



 


dme-miR-33
561


aggugcauuguagucgcauug



 


dme-miR-280
562


uguauuuacguugcauaugaaaugaua



 


dme-miR-281-1*
563


aagagagcuguccgucgacagu



 


dme-miR-281
564


ugucauggaauugcucucuuugu



 


dme-miR-282
565


aaucuagccucuacuaggcuuugucugu



 


dme-miR-283
566


uaaauaucagcugguaauucu



 


dme-miR-284
567


ugaagucagcaacuugauuccagcaauug



 


dme-miR-281-2*
568


aagagagcuauccgucgacagu



 


dme-miR-34
569


uggcagugugguuagcugguug



 


dme-miR-124
570


uaaggcacgcggugaaugccaag



 


dme-miR-79
571


uaaagcuagauuaccaaagcau



 


dme-miR-276b*
572


cagcgagguauagaguuccuacg



 


dme-miR-276b
573


uaggaacuuaauaccgugcucu



 


dme-miR-210
574


uugugcgugugacagcggcua



 


dme-miR-285
575


uagcaccauucgaaaucagugc



 


dme-miR-100
576


aacccguaaauccgaacuugug



 


dme-miR-92b
577


aauugcacuagucccggccugc



 


dme-miR-286
578


ugacuagaccgaacacucgugcu



 


dme-miR-287
579


uguguugaaaaucguuugcac



 


dme-miR-87
580


uugagcaaaauuucaggugug



 


dme-miR-263b
581


cuuggcacugggagaauucac



 


dme-miR-288
582


uuucaugucgauuucauuucaug



 


dme-miR-289
583


uaaauauuuaaguggagccugcgacu



 


dme-bantam
584


ugagaucauuuugaaagcugauu



 


dme-miR-303
585


uuuagguuucacaggaaacuggu



 


dme-miR-31b
586


uggcaagaugucggaauagcug



 


dme-miR-304
587


uaaucucaauuuguaaaugugag



 


dme-miR-305
588


auuguacuucaucaggugcucug



 


dme-miR-9c
589


ucuuugguauucuagcuguaga



 


dme-miR-306
590


ucagguacuuagugacucucaa



 


dme-miR-306*
591


gggggucacucugugccugugc



 


dme-miR-9b
592


ucuuuggugauuuuagcuguaug



 


dme-let-7
593


ugagguaguagguuguauagu



 


dme-miR-125
594


ucccugagacccuaacuuguga



 


dme-miR-307
595


ucacaaccuccuugagugag



 


dme-miR-308
596


aaucacaggauuauacugugag



 


dme-miR-31a
597


uggcaagaugucggcauagcuga



 


dme-miR-309
598


gcacuggguaaaguuuguccua



 


dme-miR-310
599


uauugcacacuucccggccuuu



 


dme-miR-311
600


uauugcacauucaccggccuga



 


dme-miR-312
601


uauugcacuugagacggccuga



 


dme-miR-313
602


uauugcacuuuucacagcccga



 


dme-miR-314
603


uauucgagccaauaaguucgg



 


dme-miR-315
604


uuuugauuguugcucagaaagc



 


dme-miR-316
605


ugucuuuuuccgcuuacuggcg



 


dme-miR-317
606


ugaacacagcuggugguauccagu



 


dme-miR-318
607


ucacugggcuuuguuuaucuca



 


dme-miR-2c
608


uaucacagccagcuuugaugggc



 


dme-miR-iab-4-5p
609


acguauacugaauguauccuga



 


dme-miR-iab-4-3p
610


cgguauaccuucaguauacguaac



 


rno-miR-322
611


aaacaugaagcgcugcaaca



 


rno-miR-323
612


gcacauuacacggucgaccucu



 


rno-miR-301
613


cagugcaauaguauugucaaagcau



 


rno-miR-324-5p
614


cgcauccccuagggcauuggugu



 


rno-miR-324-3p
615


ccacugccccaggugcugcugg



 


rno-miR-325
616


ccuaguaggugcucaguaagugu



 


rno-miR-326
617


ccucugggcccuuccuccagu



 


rno-let-7d
618


agagguaguagguugcauagu



 


rno-let-7d*
619


cuauacgaccugcugccuuucu



 


rno-miR-328
620


cuggcccucucugcccuuccgu



 


rno-miR-329
621


aacacacccagcuaaccuuuuu



 


rno-miR-330
622


gcaaagcacagggccugcagaga



 


rno-miR-331
623


gccccugggccuauccuagaa



 


rno-miR-333
624


guggugugcuaguuacuuuu



 


rno-miR-140
625


agugguuuuacccuaugguag



 


rno-miR-140*
626


uaccacaggguagaaccacggaca



 


rno-miR-336
627


ucacccuuccauaucuagucu



 


rno-miR-337
628


uucagcuccuauaugaugccuuu



 


rno-miR-148b
629


ucagugcaucacagaacuuugu



 


rno-miR-338
630


uccagcaucagugauuuuguuga



 


rno-miR-339
631


ucccuguccuccaggagcuca



 


rno-miR-341
632


ucgaucggucggucggucagu



 


rno-miR-342
633


ucucacacagaaaucgcacccguc



 


rno-miR-344
634


ugaucuagccaaagccugaccgu



 


rno-miR-345
635


ugcugaccccuaguccagugc



 


rno-miR-346
636


ugucugccugagugccugccucu



 


rno-miR-349
637


cagcccugcugucuuaaccucu



 


rno-miR-129
638


cuuuuugcggucugggcuugcu



 


rno-miR-129*
639


aagcccuuaccccaaaaagcau



 


rno-miR-20
640


uaaagugcuuauagugcagguag



 


rno-miR-20*
641


acugcauuacgagcacuuaca



 


rno-miR-350
642


uucacaaagcccauacacuuucac



 


rno-miR-7
643


uggaagacuagugauuuuguu



 


rno-miR-7*
644


caacaaaucacagucugccaua



 


rno-miR-351
645


ucccugaggagcccuuugagccug



 


rno-miR-135b
646


uauggcuuuucauuccuaugug



 


rno-miR-151*
647


ucgaggagcucacagucuagua



 


rno-miR-151
648


acuagacugaggcuccuugagg



 


rno-miR-101b
649


uacaguacugugauagcugaag



 


rno-let-7a
650


ugagguaguagguuguauaguu



 


rno-let-7b
651


ugagguaguagguugugugguu



 


rno-let-7c
652


ugagguaguagguuguaugguu



 


rno-let-7e
653


ugagguaggagguuguauagu



 


rno-let-7f
654


ugagguaguagauuguauaguu



 


rno-let-7i
655


ugagguaguaguuugugcu



 


rno-miR-7b
656


uggaagacuugugauuuuguu



 


rno-miR-9
657


ucuuugguuaucuagcuguauga



 


rno-miR-10a
658


uacccuguagauccgaauuugug



 


rno-miR-10b
659


uacccuguagaaccgaauuugu



 


rno-miR-15b
660


uagcagcacaucaugguuuaca



 


rno-miR-16
661


uagcagcacguaaauauuggcg



 


rno-miR-17
662


caaagugcuuacagugcagguagu



 


rno-miR-18
663


uaaggugcaucuagugcagaua



 


rno-miR-19b
664


ugugcaaauccaugcaaaacuga



 


rno-miR-19a
665


ugugcaaaucuaugcaaaacuga



 


rno-miR-21
666


uagcuuaucagacugauguuga



 


rno-miR-22
667


aagcugccaguugaagaacugu



 


rno-miR-23a
668


aucacauugccagggauuucc



 


rno-miR-23b
669


aucacauugccagggauuaccac



 


rno-miR-24
670


uggcucaguucagcaggaacag



 


rno-miR-25
671


cauugcacuugucucggucuga



 


rno-miR-26a
672


uucaaguaauccaggauaggcu



 


rno-miR-26b
673


uucaaguaauucaggauagguu



 


rno-miR-27b
674


uucacaguggcuaaguucug



 


rno-miR-27a
675


uucacaguggcuaaguuccgc



 


rno-miR-28
676


aaggagcucacagucuauugag



 


rno-miR-29b
677


uagcaccauuugaaaucagugu



 


rno-miR-29a
678


cuagcaccaucugaaaucgguu



 


rno-miR-29c
679


uagcaccauuugaaaucgguua



 


rno-miR-30c
680


uguaaacauccuacacucucagc



 


rno-miR-30e
681


uguaaacauccuugacugga



 


rno-miR-30b
682


uguaaacauccuacacucagc



 


rno-miR-30d
683


uguaaacauccccgacuggaag



 


rno-miR-30a
684


cuuucagucggauguuugcagc



 


rno-miR-31
685


aggcaagaugcuggcauagcug



 


rno-miR-32
686


uauugcacauuacuaaguugc



 


rno-miR-33
687


gugcauuguaguugcauug



 


rno-miR-34b
688


uaggcaguguaauuagcugauug



 


rno-miR-34c
689


aggcaguguaguuagcugauugc



 


rno-miR-34a
690


uggcagugucuuagcugguuguu



 


rno-miR-92
691


uauugcacuugucccggccug



 


rno-miR-93
692


caaagugcuguucgugcagguag



 


rno-miR-96
693


uuuggcacuagcacauuuuugcu



 


rno-miR-98
694


ugagguaguaaguuguauuguu



 


rno-miR-99a
695


aacccguagauccgaucuugug



 


rno-miR-99b
696


cacccguagaaccgaccuugcg



 


rno-miR-100
697


aacccguagauccgaacuugug



 


rno-miR-101
698


uacaguacugugauaacugaag



 


rno-miR-103
699


agcagcauuguacagggcuauga



 


rno-miR-106b
700


uaaagugcugacagugcagau



 


rno-miR-107
701


agcagcauuguacagggcuauca



 


rno-miR-122a
702


uggagugugacaaugguguuugu



 


rno-miR-124a
703


uuaaggcacgcggugaaugcca



 


rno-miR-125a
704


ucccugagacccuuuaaccugug



 


rno-miR-125b
705


ucccugagacccuaacuuguga



 


rno-miR-126*
706


cauuauuacuuuugguacgcg



 


rno-miR-126
707


ucguaccgugaguaauaaugc



 


rno-miR-127
708


ucggauccgucugagcuuggcu



 


rno-miR-128a
709


ucacagugaaccggucucuuuu



 


rno-miR-128b
710


ucacagugaaccggucucuuuc



 


rno-miR-130a
711


cagugcaauguuaaaagggc



 


rno-miR-130b
712


cagugcaaugaugaaagggcau



 


rno-miR-132
713


uaacagucuacagccauggucg



 


rno-miR-133a
714


uugguccccuucaaccagcugu



 


rno-miR-134
715


ugugacugguugaccagaggg



 


rno-miR-135a
716


uauggcuuuuuauuccuauguga



 


rno-miR-136
717


acuccauuuguuuugaugaugga



 


rno-miR-137
718


uauugcuuaagaauacgcguag



 


rno-miR-138
719


agcugguguugugaauc



 


rno-miR-139
720


ucuacagugcacgugucu



 


rno-miR-141
721


aacacugucugguaaagaugg



 


rno-miR-142-5p
722


cauaaaguagaaagcacuac



 


rno-miR-142-3p
723


uguaguguuuccuacuuuaugga



 


rno-miR-143
724


ugagaugaagcacuguagcuca



 


rno-miR-144
725


uacaguauagaugauguacuag



 


rno-miR-145
726


guccaguuuucccaggaaucccuu



 


rno-miR-146
727


ugagaacugaauuccauggguu



 


rno-miR-150
728


ucucccaacccuuguaccagug



 


rno-miR-152
729


ucagugcaugacagaacuugg



 


rno-miR-153
730


uugcauagucacaaaaguga



 


rno-miR-154
731


uagguuauccguguugccuucg



 


rno-miR-181c
732


aacauucaaccugucggugagu



 


rno-miR-181a
733


aacauucaacgcugucggugagu



 


rno-miR-181b
734


aacauucauugcugucgguggguu



 


rno-miR-183
735


uauggcacugguagaauucacug



 


rno-miR-184
736


uggacggagaacugauaagggu



 


rno-miR-185
737


uggagagaaaggcaguuc



 


rno-miR-186
738


caaagaauucuccuuuugggcuu



 


rno-miR-187
739


ucgugucuuguguugcagccg



 


rno-miR-190
740


ugauauguuugauauauuaggu



 


rno-miR-191
741


caacggaaucccaaaagcagcu



 


rno-miR-192
742


cugaccuaugaauugacagcc



 


rno-miR-193
743


aacuggccuacaaagucccag



 


rno-miR-194
744


uguaacagcaacuccaugugga



 


rno-miR-195
745


uagcagcacagaaauauuggc



 


rno-miR-196
746


uagguaguuucauguuguugg



 


rno-miR-199a
747


cccaguguucagacuaccuguuc



 


rno-miR-200c
748


aauacugccggguaaugaugga



 


rno-miR-200a
749


uaacacugucugguaacgaugu



 


rno-miR-200b
750


cucuaauacugccugguaaugaug



 


rno-miR-203
751


gugaaauguuuaggaccacuag



 


rno-miR-204
752


uucccuuugucauccuaugccu



 


rno-miR-205
753


uccuucauuccaccggagucug



 


rno-miR-206
754


uggaauguaaggaagugugugg



 


rno-miR-208
755


auaagacgagcaaaaagcuugu



 


rno-miR-210
756


cugugcgugugacagcggcug



 


rno-miR-211
757


uucccuuugucauccuuugccu



 


rno-miR-212
758


uaacagucuccagucacggcc



 


rno-miR-213
759


accaucgaccguugauuguacc



 


rno-miR-214
760


acagcaggcacagacaggcag



 


rno-miR-216
761


uaaucucagcuggcaacugug



 


rno-miR-217
762


uacugcaucaggaacugacuggau



 


rno-miR-218
763


uugugcuugaucuaaccaugu



 


rno-miR-219
764


ugauuguccaaacgcaauucu



 


rno-miR-221
765


agcuacauugucugcuggguuuc



 


rno-miR-222
766


agcuacaucuggcuacugggucuc



 


rno-miR-223
767


ugucaguuugucaaauacccc



 


rno-miR-290
768


cucaaacuaugggggcacuuuuu



 


rno-miR-291-5p
769


caucaaaguggaggcccucucu



 


rno-miR-291-3p
770


aaagugcuuccacuuugugugcc



 


rno-miR-292-5p
771


acucaaacugggggcucuuuug



 


rno-miR-292-3p
772


aagugccgccagguuuugagugu



 


rno-miR-296
773


agggcccccccucaauccugu



 


rno-miR-297
774


auguaugugugcauguaugcaug



 


rno-miR-298
775


ggcagaggagggcuguucuucc



 


rno-miR-299
776


ugguuuaccgucccacauacau



 


rno-miR-300
777


uaugcaagggcaagcucucuuc



 


rno-miR-320
778


aaaagcuggguugagagggcgaa



 


rno-miR-321
779


uaagccagggauuguggguuc









Although the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it will be appreciated that various changes and modifications may be made without departing from the teachings herein. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings herein.

## Claims

1. A kit comprising:
a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target-specific portion that is complementary to a target polynucleotide and is configured to be extended to form an extension reaction product complementary to the target polynucleotide; and
a detector probe, wherein at least a portion of the detector probe corresponds with the stem of the linker probe, wherein the detector probe is a different molecule from the linker probe, and wherein the detector probe comprises a detectable label.

2. The kit according to claim 1, further comprising a primer pair.

3. The kit according to claim 1, further comprising a universal reverse primer, wherein at least a portion of the universal reverse primer corresponds with a region of the loop of the linker probe.

4. The kit according to claim 3, further comprising a forward primer specific for the target polynucleotide.

5. The kit according to claim 1 comprising a plurality of primer pairs, wherein each primer pair is in one reaction vessel of a plurality of reaction vessels.

6. The kit according to claim 1, wherein the detector probe further corresponds with at least a portion of the 3′ end region of the target polynucleotide.

7. The kit according to claim 1, wherein the target polynucleotide is a microRNA.

8. A kit comprising:
a linker probe, wherein the linker probe comprises a stem, a loop, and a 3′ target-specific portion that is complementary to a target polynucleotide and is configured to be extended to form an extension reaction product complementary to the target polynucleotide; and
a universal reverse primer, wherein at least a portion of the universal reverse primer corresponds with a region of the loop of the linker probe.

9. The kit according to claim 8 comprising a primer pair.

10. The kit according to claim 8, further comprising a forward primer specific for the target polynucleotide.

11. The kit according to claim 10, further comprising a detector probe, wherein at least a portion of the detector probe corresponds with the stem of the linker probe, wherein the detector probe is a different molecule from the linker probe, and wherein the detector probe comprises a detectable label.

12. The kit according to claim 8 comprising a plurality of primer pairs, wherein each primer pair is in one reaction vessel of a plurality of reaction vessels.

13. The kit according to claim 8, wherein the universal reverse primer further comprises a tail region not corresponding to the linker probe.

14. The kit according to claim 8, wherein the target polynucleotide is a microRNA.

15. An unlabeled linker probe comprising a stem, a loop, and a 3′ target-specific portion that is complementary to a target polynucleotide and is configured to be extended to form an extension reaction product complementary to the target polynucleotide.

16. The linker probe according to claim 15, wherein the probe further comprises an identifying portion.

17. The linker probe according to claim 15, wherein the stem of the linker probe comprises 12-16 base pairs.

18. The linker probe according to claim 15, wherein the 3′ target-specific portion of the linker probe comprises 6-8 nucleotides.

19. The linker probe according to claim 15, wherein at least the five 3′-most nucleotides of the 3′ target-specific portion are complementary to the target polynucleotide.

20. The linker probe according to claim 15, wherein the target polynucleotide is a microRNA.