# Patent

## Title
High throughput methods relating to microRNA expression analysis

## Abstract

The invention relates to methods and compositions for microRNA expression analysis using microarrays.

## Background
PRIORITY CLAIM
This application claims priority from U.S. Provisional Application Ser. No. 60/584,381, filed Jun. 30, 2004, and from U.S. Provisional Application Ser. No. 60/607,531, filed Sep. 7, 2004, both of which are incorporated herein by reference.


BACKGROUND OF THE INVENTION
The invention relates to methods and compositions for microRNA expression analysis using microarrays.
MicroRNAs are a new class of small regulatory RNAs that are found in a variety of organisms, including nematodes, plants, insects and mammals. In invertebrates, microRNAs have been implicated as regulators of developmental timing, neuronal differentiation, cell proliferation, programmed cell death, and fat metabolism. In C. elegans, lin-4 and let-7 act in developmental timing, and the microRNA lsy-6 controls neuronal asymmetry. In Drosophila, the microRNAs bantam and mir-14 act in the regulation of cell growth, spermatogenesis and cell death. The mouse microRNA miR-181 functions in hematopoietic differentiation, and two human microRNAs are involved in chronic lymphocytic leukemia, the most common form of adult leukemia in the western world.
Mature microRNAs are excised from a stem-loop precursor that itself can be transcribed as part of a longer primary RNA (pri-miRNA). The pri-miRNA appears to be processed by the RNAse Drosha in the nucleus, cleaving the RNA at the base of the stem-loop. This cut defines one end of the microRNA. The precursor microRNA is then exported by Ran-GTP and Exportin-5 to the cytoplasm, where it is further processed by the RNAse Dicer, which recognizes the stem portion of the microRNA and cleaves both strands about 22 nucleotides from the base of the stem. The two strands of the resulting dsRNA are differentially stable, and the mature microRNA resides on the strand that is more stable. Mature microRNAs can be found associated with the proteins eIF2C2 (an Argonaute-like protein), Gemin2, and Gemin3 and are thought to act in a protein-RNA complex with these and maybe other proteins.
Most animal microRNAs inhibit the protein expression of their target gene. Typically, the target gene encodes an mRNA that contains a sequence in its 3′UTR that is partially complementary to the corresponding microRNA. While some plant microRNAs also function in this way, most plant microRNAs cause the cleavage of target mRNAs at sites that are perfectly complementary to the microRNAs.
More than 200 microRNAs are encoded by the human genome. Few of these microRNAs have been characterized. To date, the function of individual microRNAs has been analyzed using time-intensive procedures, such as dot-blot and northern blotting analysis, techniques that require the isolation of large amounts of RNA. A need exists for a high-throughput method that allows for the simultaneous analysis of multiple microRNAs and that provides for the analysis of microRNA expression when only small amounts of starting material are available.
SUMMARY OF THE INVENTION
In general, the invention relates to methods and compositions for microRNA expression analysis using microarrays. As described in more detail below, these methods allow for the detection of all known microRNAs of a given species in parallel. Unlike existing methods of mRNA analysis, the methods described herein optionally provide for the amplification of microRNAs. This amplification step facilitates the analysis of a wide range of biological materials, including small quantities of biological samples containing a limited amount of RNA.
In addition, existing methods fail to provide methods suitable for labeling RNAs as small as microRNAs. To address this need, we disclose herein a method to detectably label small RNAS. The method includes the following steps. First, small RNAs (e.g., 18-26 nucleotides) are size-selected from total RNA, for example, by using denaturing polyacrylamide gel electrophoresis. Oligonucleotide linkers (e.g., DNA, RNA, RNA/DNA hybrid, or having a block at the 3′ end to inhibit self ligation) are attached to the 5′ and 3′ ends of the small RNAs. These linkers are at least 5, 10, 12, 15, 18, 20, or 25 nucleotides in length. Such linkers optionally include sites that facilitate subsequent cloning (e.g., restriction sites), sites that promote transcription (e.g., T7 site), or sites that facilitate the purification of the microRNA (e.g., a biotin). These ligation products are optionally used as templates for amplification (e.g., RT-PCR reaction with 10 cycles of amplification). The sense-strand PCR primer contains a detectable label (e.g., a fluorescent label, an enzyme, radiolabel, or other detectable group). Binding of the detectably labeled microRNA to an oligonucleotide that is at least partially complementary to the microRNA is determined using standard techniques based on a characteristic of the detectable group such as its enzyme activity, radioactivity, or fluorescence. In one working embodiment, a Cy3 fluorophore is attached to the PCR primer's 5′ end, thereby fluorescently labeling the sense strand of the PCR product. The PCR product is then denatured and hybridized to a microarray. In contrast to existing methods that rely on substantial quantities of starting material, when the microRNA is amplified prior to hybridization, relatively low amounts of starting material may be used. Thus, the present invention is particularly advantageous for analyzing microRNA expression in a biological sample (e.g., biopsy specimen) isolated from a patient.
While the detectably labeled microRNA may be hybridized to any complementary oligonucleotide, it is preferably hybridized to a microarray that includes 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, 500, 750, or 1000 oligonucleotides. Some of these oligonucleotides are complementary to the detectably labeled microRNAs. Optionally, at least one or more negative control oligonucleotides are included that fail to bind a microRNA. Optionally, at least one or more positive control probes are included that bind a microRNA. Such probes contain a means for affixing the probe to a solid substrate (e.g., a membrane, glass slide, bead). Optionally, this means is a free amine group at the 5′ terminus that allows the probe to be attached (e.g., printed) onto an amine-binding glass slides. The probes are covalently linked to the glass surface. In some examples, the probes are affixed to the substrate in duplicate, triplicate, or quadruplicate. Hybridization is carried out at any temperature that optimizes binding sensitivity and specificity, i.e, a temperature that allows the detectably labeled microRNA to specifically bind to an oligonucleotide that is at least partially complementary to the microRNA. Preferably, a hybridization temperature between 40° C. and 60° C. is selected, more preferably between 45° C. and 55° C. (e.g., 47° C., 48° C., 49° C., 51° C., 53° C.), and most preferably the hybridization is carried out at 50° C.
In a first aspect, the invention generally features a method for identifying microRNA expression in a sample. The method includes providing a microRNA isolated from a sample; appending at least one linker to the microRNA; detectably labeling the microRNA; contacting a microarray comprising at least 2 oligonucleotides with the detectably labeled microRNA; and detecting binding of the detectably labeled microRNA to the microarray.
In another aspect, the invention features a method for identifying microRNA expression in a sample, the method includes providing a microRNA isolated from a sample; amplifying the microRNA to produce a detectably labeled microRNA; contacting a microarray comprising at least 2 oligonucleotides with the detectably labeled microRNA; and detecting binding of the detectably labeled microRNA to the microarray.
In another aspect, the invention features a method for identifying differential expression of a microRNA in a test sample. The method includes providing a microRNA isolated from the test sample; appending at least one linker to the microRNA; detectably labeling the microRNA; contacting a microarray comprising at least 2 microRNAs with the detectably labeled microRNA; and detecting a difference in the binding of the detectably labeled microRNA to the microarray relative to the binding of a corresponding control sample.
In another aspect, the invention features a method for identifying differential expression of a microRNA in a test sample. The method includes providing a microRNA isolated from a test sample; amplifying the microRNA to produce a detectably labeled microRNA; contacting a microarray comprising at least 2 microRNAs with the detectably labeled microRNA; and detecting a difference in the binding of the detectably labeled microRNA to the microarray relative to the binding of a corresponding control sample.
In some embodiments of the above aspects, the test sample is a tissue sample from a subject having a disease, condition, or disorder selected from the group consisting of autoinflammatory disorders (e.g., asthma, allergic intraocular inflammatory diseases, arthritis, atopic dermatitis, atopic eczema, diabetes, haemolytic anaemia, inflammatory dermatoses, inflammatory bowel or gastrointestinal disorders, multiple sclerosis, myasthenia gravis, pruritis/inflammation, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus), proliferative diseases (e.g., leukemias, lymphomas, sarcomas and carcinomas), cardiovascular diseases (e.g., atherosclerosis, hypertension, cardiac artery disease, myocardial infarction, or congestive heart failure), obesity, or an obesity related diseases (e.g., diabetes).
In another aspect, the invention features a method of diagnosing a subject as having, or having a propensity to develop, a microRNA-related disorder. The method includes providing a microRNA isolated from a cell of the subject; appending at least one linker to the microRNA or amplifying the microRNA from the subject; detectably labeling the microRNA; and determining the level of expression of the microRNA, where an alteration in the level of expression of the microRNA relative to a reference, indicates that the patient has or has a propensity to develop a microRNA-related disorder.
In another aspect, the invention features a method for producing a detectably labeled microRNA. The method includes providing an isolated microRNA, and attaching a linker bound to a to detectable label to the microRNA.
In yet another aspect, the invention features a method for producing a detectably labeled microRNA. The method includes amplifying a microRNA from a sample, and detectably labeling the microRNA.
In a related aspect, the invention features a detectably labeled microRNA produced according to the methods of any of the above aspects.
In another aspect, the invention features a method for producing a microRNA microarray. The method includes providing a microRNA; appending at least one linker to the microRNA; and affixing the detectably labeled microRNAs to a solid support.
In yet another aspect, the invention features a method for producing a microRNA microarray, the method includes providing at least 2, 5, 10, 25, 50, 75, 100, 125, 150, 175, 200 microRNAs; amplifying the microRNAs; and affixing the microRNAs to a solid support (e.g., a bead or a glass slide). In some embodiments, the microRNAs contain a detectable label. In other embodiments, the bead has a characteristic that provides for its identification (e.g., fluorophore, size, color, charge, or any other identifiable signal or modification).
In yet another aspect, the invention features a method of microRNA hybridization. The method includes contacting a microRNA probe and a target nucleic acid at a temperature between 40° and 60° C., 45° C. and 55° C., or at 50° C. under conditions suitable for binding.
In another aspect, the invention features a kit for microRNA expression analysis. The kit contains at least 2 detectably labeled microRNAs produced according to any method of a previous aspect, where the microRNAs are bound to a substrate and the kit further contains directions for the use of the detectably labeled microRNAs for the detection of microRNA expression.
In various embodiments of any of the above aspects, the linker contains oligonucleotides (e.g., RNA, DNA, or is an RNA/DNA hybrid). In other embodiments, one or two linkers are appended in a ligation reaction. In another embodiment, the microRNA is useful as a template for a reverse transcriptase polymerase chain reaction (RT-PCR). In yet another embodiment, the microRNA is detectably labeled during the performance of the polymerase chain reaction (PCR). In another embodiment, the linker contains a T7 promoter, at least one restriction site, or a modification that facilitates purification of the microRNA (e.g., biotin).
In various embodiments of any of the above aspects, the microRNA is detectably labeled during the performance of PCR. In some embodiments, the detectable label is a fluorophore. In other embodiments of the above aspects, the detectable label is detected by analyzing enzyme activity, by direct immunoassay, or by a radiometric assay. In still other embodiments, the sample is a tissue sample (e.g., a neoplastic tissue sample). In yet other embodiments, the microarray contains at least 10, 25, 50, 75, or 100 oligonucleotides. In other embodiments of any of the above aspects, the microRNA is one of those listed in Table 1 or 2, or disclosed herein.
In another aspect, the invention features a microarray containing at least two nucleic acid molecules, or fragments thereof, which are regulated in the developing rat brain, bound to a solid support, where at least 90% of the nucleic acid molecules on the support are selected from any one or more of the group consisting of rno-miR-b, rno-let-7a, rno-let-7b, rno-let-7c, rno-let-7d, rno-let-7i, rno-miR-7, rno-miR-9, rno-miR-16, rno-miR-17-5p, rno-miR-24, rno-miR-26b, rno-miR-28, rno-miR-29a, rno-miR-29b, rno-miR-29c, rno-miR-30b, rno-miR-30c, rno-miR-92, rno-miR-93, rno-miR-99a, rno-miR-99b, rno-miR-101b, rno-miR-103, rno-miR-124a, rno-miR-125a, rno-miR-125b, rno-miR-127, rno-miR-128a, rno-miR-128a or b, rno-miR-128b, rno-miR-129, rno-miR-130a, rno-miR-132, rno-miR-136, rno-miR-138, rno-miR-139, rno-miR-140*, rno-miR-142-3p, rno-miR-145, rno-miR-146, rno-miR-150, rno-miR-154, rno-miR-185, rno-miR-191, rno-miR-213, rno-miR-300, rno-miR-323, rno-miR-324, rno-miR-325, rno-miR-338, rno-miR-342, and rno-miR-345, or any nucleic acid molecule listed in Table 2.
In another aspect, the invention features a purified nucleic acid library containing at least two nucleic acid molecules that are regulated in the developing rat brain selected from any one or more of the group consisting of rno-miR-b, rno-let-7a, rno-let-7b, rno-let-7c, rno-let-7d, rno-let-7i, rno-miR-7, rno-miR-9, rno-miR-16, rno-miR-17-5p, rno-miR-24, rno-miR-26b, rno-miR-28, rno-miR-29a, rno-miR-29b, rno-miR-29c, rno-miR-30b, rno-miR-30c, rno-miR-92, rno-miR-93, rno-miR-99a, rno-miR-99b, rno-miR-101b, rno-miR-103, rno-miR-124a, rno-miR-125a, rno-miR-125b, rno-miR-127, rno-miR-128a, rno-miR-128a or b, rno-miR-128b, rno-miR-129, rno-miR-130a, rno-miR-132, rno-miR-136, rno-miR-138, rno-miR-139, rno-miR-140*, rno-miR-142-3p, rno-miR-145, rno-miR-146, rno-miR-150, rno-miR-154, rno-miR-185, rno-miR-191, rno-miR-213, rno-miR-300, rno-miR-323, rno-miR-324, rno-miR-325, rno-miR-338, rno-miR-342, and rno-miR-345 or a nucleic acid molecule listed in Table 2.
In yet another aspect, the invention features a microarray comprising at least two nucleic acid molecules, or fragments thereof, that are regulated in the developing rat brain and bound to a solid support, where at least 90% of the nucleic acid molecules on the support are selected from any one or more of the group consisting of mml-let-7a, mml-let-7a or c, mml-let-7b, mml-let-7c, mml-let-7d, mml-let-7e, mml-let-7f, mml-let-7g, mml-let-7i, mml-miR-7-1, mml-miR-9, mml-miR-16, mml-miR-17-5p, mml-miR-26a, mml-miR-30b, mml-miR-30c, mml-miR-33, mml-miR-92, mml-miR-99a, mml-miR-99b, mml-mir-100, mml-miR-103, mml-miR-103 or 107, mml-miR-124a, mml-miR-124a, mml-miR-125a, mml-miR-125b, mml-miR-126, mml-miR-126*, mml-miR-128a, mml-miR-128a or b, mml-miR-128b, mml-miR-129-2, mml-miR-136, mml-miR-137, mml-miR-140, mml-miR-145, mml-miR-149, mml-miR-181a or 213, mml-miR-181b, mml-miR-181c, mml-miR-185, mml-miR-195, and mml-miR-221.
In yet another aspect, the invention features a purified nucleic acid library containing at least two nucleic acid molecules regulated in the developing rat brain and selected from any one or more of the group consisting of mml-let-7a, mml-let-7a or c, mml-let-7b, mml-let-7c, mml-let-7d, mml-let-7e, mml-let-7f, mml-let-7g, mml-let-7i, mml-miR-7-1, mml-miR-9, mml-miR-16, mml-miR-17-5p, mml-miR-26a, mml-miR-30b, mml-miR-30c, mml-miR-33, mml-miR-92, mml-miR-99a, mml-miR-99b, mml-mir-100, mml-miR-103, mml-miR-103 or 107, mml-miR-124a, mml-miR-124a, mml-miR-125a, mml-miR-125b, mml-miR-126, mml-miR-126*, mml-miR-128a, mml-miR-128a or b, mml-miR-128b, mml-miR-129-2, mml-miR-136, mml-miR-137, mml-miR-140, mml-miR-145, mml-miR-149, mml-miR-181a or 213, mml-miR-181b, mml-miR-181c, mml-miR-185, mml-miR-195, and mml-miR-221, or any of the nucleic acid molecules listed in Table 2.
By “cell” is meant a single-cellular organism, cell from a multi-cellular organism, or cell contained in a multi-cellular organism.
By “differentially expressed” is meant a difference in the expression level of a nucleic acid or polypeptide in a test sample relative to the level of the nucleic acid in a control sample or relative or other reference. This difference may be either an increase or a decrease in expression, when compared to control conditions. Preferably, the increase or decrease is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%.
By “duplex” is meant a single unit containing paired sense and antisense domains.
By “hybridize” is meant pair to form a duplex or double-stranded complex containing complementary paired nucleobase sequences, or portions thereof. Preferably, hybridization occurs under physiological conditions, or under various conditions of stringency. (See, e.g., Wahl and Berger Methods Enzymol. 152:399, 1987; Kimmel, A. R. Methods Enzymol. 152:507, 1987). For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and most preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc Natl Acad Sci USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. Typically, complementary nucleobases hybridize via hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes, which, in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By “linker” is meant a short segment of synthetic oligomer that acts as a connecting element. Such elements are typically used in connecting longer nucleic acid segments.
By “microRNA” is meant a small non-coding RNA. Typically, microRNAs are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides in length and are processed from a precursor RNA containing a stem-loop.
By “microRNA-related disorder” is meant a pathological condition associated with an alteration in the sequence, expression, or biological activity of a microRNA. In one example, a microRNA-related disorder is a neoplasm characterized as having a decrease in the expression of a microRNA.
By “microarray” is meant an organized collection of at least two nucleic acid molecules affixed to a solid support. In some embodiments, a microRNA microarray is composed of oligonucleotides having at least a portion (e.g., 10, 15, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides) of two or more nucleic acid sequences listed in Table 1 or 2. A microarray contains at least 2, 5, 10, 25, 50, 75, 100, 150, 200, 250, or 300 nucleic acid molecule members.
By “portion” is meant a fragment of a protein or nucleic acid that is substantially identical to a reference protein or nucleic acid. In some embodiments the portion retains at least 50% 75%, or 80%, or more preferably 90%, 95%, or even 99% of the biological activity of the reference protein or nucleic acid described herein.
By “operably linked” is meant that a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide.
By “proliferative disease” is meant a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancer is an example of a proliferative disease. Examples of cancers include, without limitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).
By “positioned for expression” is meant that the polynucleotide of the invention (e.g., a DNA molecule) is positioned adjacent to a DNA sequence that directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a recombinant polypeptide of the invention, or an RNA molecule).
By “promoter” is meant a polynucleotide sufficient to direct transcription.
By “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation).
By “reporter gene” is meant a gene encoding a polypeptide whose expression may be assayed; such polypeptides include, without limitation, glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), and beta-galactosidase.
The invention features methods and compositions relating to the analysis of microRNAs. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 A is an alignment of the top BLAST hit for each mouse microRNA precursor sequence (Rfam, Release 3.0) with the corresponding rat genomic sequence (public release draft genome assembly, version 3.1) (SEQ ID NOS:227-334). FIG. 1A shows mouse let-7a- 1 (SEQ ID NO:227) and the corresponding rat sequence (SEQ ID NO:228); mouse let-7a-2 (SEQ ID NO:229) and the corresponding rat sequence (SEQ ID NO:230); mouse let-7b (SEQ ID NO: 231) and the corresponding rat sequence (SEQ ID NO: 232); mouse let-7c-1(SEQ ID NO: 233) and the corresponding rat sequence (SEQ ID NO:234); mouse let-7c-2 (SEQ ID NO:235) and the corresponding rat sequence (SEQ ID NO:236); mouse let-7e (SEQ ID NO:237) and the corresponding rat sequence (SEQ ID NO:238); mouse let-7i (SEQ ID NO:239) and the corresponding rat sequence (SEQ ID NO:240); mouse miR-7-1 (SEQ ID NO:241) and the corresponding rat sequence (SEQ ID NO:242); mouse miR-7-2 (SEQ ID NO:243) and the corresponding rat sequence (SEQ ID NO:244); mouse miR-9-1 (SEQ ID NO:245) and the corresponding rat sequence (SEQ ID NO:246); mouse miR-9-3 (SEQ ID NO:247) and the corresponding rat sequence (SEQ ID NO:248); mouse miR-9-2 (SEQ ID NO:249) and the corresponding rat sequence (SEQ ID NO:250); mouse miR-16-2 (SEQ ID NO:251) and the corresponding rat sequence (SEQ ID NO:252); mouse miR-24-1(SEQ ID NO:253) and the corresponding rat sequence (SEQ ID NO:254); mouse miR-24-2 (SEQ ID NO:255) and the corresponding rat sequence (SEQ ID NO:256); mouse miR-26b (SEQ ID NO:257) and the corresponding rat sequence (SEQ ID NO:258); mouse miR-28 (SEQ ID NO:259) and the corresponding rat sequence (SEQ ID NO:260); mouse miR-29a (SEQ ID NO:261) and the corresponding rat sequence (SEQ ID NO:262); mouse miR-29c (SEQ ID NO:263) and the corresponding rat sequence (SEQ ID NO:264); mouse miR-29b-1(SEQ ID NO:265) and the corresponding rat sequence (SEQ ID NO:266); mouse miR-29b-2 (SEQ ID NO:267) and the corresponding rat sequence (SEQ ID NO:268); mouse miR-30b (SEQ ID NO:269) and the corresponding rat sequence (SEQ ID NO:270); mouse miR-30c-1(SEQ ID NO:271) and the corresponding rat sequence (SEQ ID NO:272); mouse miR-30c-2 (SEQ ID NO:273) and the corresponding rat sequence (SEQ ID NO:274); mouse miR-92-2 (SEQ ID NO:275) and the corresponding rat sequence (SEQ ID NO:276); mouse miR-93 (SEQ ID NO:277) and the corresponding rat sequence (SEQ ID NO:278); mouse miR-99a (SEQ ID NO:279) and the corresponding rat sequence (SEQ ID NO:280); mouse miR-99b (SEQ ID NO:281) and the corresponding rat sequence (SEQ ID NO:282); mouse miR-103-1 (SEQ ID NO:283) and the corresponding rat sequence (SEQ ID NO:284); mouse miR-103-2 (SEQ ID NO:285) and the corresponding rat sequence (SEQ ID NO:286); mouse miR-124a-1 (SEQ ID NO:287) and the corresponding rat sequence (SEQ ID NO:288); mouse miR-124a-2 (SEQ ID NO:289) and the corresponding rat sequence (SEQ ID NO:290); mouse miR-124a-3 (SEQ ID NO:291) and the corresponding rat sequence (SEQ ID NO:292); mouse miR-125a (SEQ ID NO:293) and the corresponding rat sequence (SEQ ID NO:294); mouse miR- 125b-2 (SEQ ID NO:295) and the corresponding rat sequence (SEQ ID NO:296); mouse miR-125b-1 (SEQ ID NO:297) and the corresponding rat sequence (SEQ ID NO:298); mouse miR-127 (SEQ ID NO:299) and the corresponding rat sequence (SEQ ID NO:300); mouse miR-128a (SEQ ID NO:301) and the corresponding rat sequence (SEQ ID NO:302); mouse miR-128b (SEQ ID NO:303) and the corresponding rat sequence (SEQ ID NO:304); mouse miR-130a (SEQ ID NO:305) and the corresponding rat sequence (SEQ ID NO:306); mouse miR-132 (SEQ ID NO:307) and the corresponding rat sequence (SEQ ID NO:308); mouse miR-136 (SEQ ID NO:309) and the corresponding rat sequence (SEQ ID NO:310); mouse miR-138-1 (SEQ ID NO:311) and the corresponding rat sequence (SEQ ID NO:312); mouse miR-138-2 (SEQ ID NO:3 13) and the corresponding rat sequence (SEQ ID NO:314); mouse miR-139 (SEQ ID NO:3 15) and the corresponding rat sequence (SEQ ID NO:316); mouse miR-142 (SEQ ID NO:317) and the corresponding rat sequence (SEQ ID NO:3 18); mouse miR-145 (SEQ ID NO:319) and the corresponding rat sequence (SEQ ID NO:320); mouse miR-146 (SEQ ID NO:321) and the corresponding rat sequence (SEQ ID NO:322); mouse miR-150 (SEQ ID NO:323) and the corresponding rat sequence (SEQ ID NO:324); mouse miR-154 (SEQ ID NO:325) and the corresponding rat sequence (SEQ ID NO:326); mouse miR-185 (SEQ ID NO:327) and the corresponding rat sequence (SEQ ID NO:328); mouse miR-191 (SEQ ID NO:329) and the corresponding rat sequence (SEQ ID NO:330;: mouse miR-213 (SEQ ID NO:331) and the corresponding rat sequence (SEQ ID NO:332); and mouse miR-300 (SEQ ID NO:333) and the corresponding rat sequence (SEQ ID NO:334). FIG. 1B shows predicted precursor secondary structures for selected rat microRNA genes (SEQ ID NOS:335-352). FIG. 1B shows the structure of rno-let-7a-2 (SEQ ID NO: 335), rno-let-7b (SEQ ID NO: 336), rno-mir-7-1 (SEQ ID NO: 337), rno-mir-7-2 (SEQ ID NO: 338), rno-mir-16-2 (SEQ ID NO: 339), rno-mir-24-2 (SEQ ID NO: 340), rno-mir-26b (SEQ ID NO: 341), rno-mir-28 (SEQ ID NO: 342), rno-mir-30b (SEQ ID NO: 343), rno-30c-2 (SEQ ID NO: 344), rno-mir-92-2 (SEQ ID NO: 345), rno-mir-93 (SEQ ID NO: 346), rno-mir-99a (SEQ ID NO: 347), rno-mir-125-b2 (SEQ ID NO: 348), rno-mir-130a (SEQ ID NO: 349), rno-mir-138-1 (SEQ ID NO: 350), mo-mir-138-2 (SEQ ID NO: 351), and rno-mir-146 (SEQ ID NO: 352). The selected rat microRNA genes have microRNA precursor sequences that differ from their corresponding mouse precursors. We used the infold algorithm to make secondary structure predictions (Zuker NucleicAcids Res, 3 1:3406-3415, 2003).
FIG. 2A shows the predicted stem-loop structure of a novel mammalian microRNA, rno-miR-B (SEQ ID NO:353). The stem-loop structure was predicted from sequences adjacent to rno-miR-B in the rat genome. The cloned (mature) sequence is shown in gray. The predicted secondary structure and the free energy calculation (ΔG, kcal/mole) were generated by the infold software (Zuker Nucleic Acids Res, 31:3406-3415, 2003). FIG. 2B shows an alignment of rat (SEQ ID NO:354), human (SEQ ID NO:355, and mouse (SEQ ID NO:356) precursor sequences for microRNA mir-B. The cloned sequence (corresponding to the mature microRNA) is shown in bold. The single mismatch is indicated by an asterix (*).
FIG. 3 depicts the predicted secondary structures for the corresponding genes from mouse (SEQ ID NO:357) and human (SEQ ID NO:358) miR-B. The cloned sequence, which corresponds to the mature microRNA, is shown in gray.
FIG. 4 shows the stem-loop precursor for smallRNA-1 (SEQ ID NO:359) from monkey. The cloned sequence, which corresponds to the mature microRNA, is shown in gray.
FIG. 5 is a photograph of a Northern blot showing that rno-miR-138 expression was restricted to brain.
FIG. 6A is a graph depicting the specificity index plotted against the calculated melting temperature for each of 23 microRNA probe pairs. Specificity was assayed using a set of 23 microRNA and mismatched probe pairs (two mismatches). The average mean spot intensities from ten independent hybridizations at 50° C. were added to give a total signal for probes corresponding to a given microRNA as well as for probes with two mismatches to the microRNA. The mismatch probe design and nucleic acid sequences are described in Table 2. The specificity index was calculated as follows: 100× (probe signal—mismatched probe signal)/probe signal. Melting temperatures for the microRNA probes were calculated using the nearest neighbors method (Breslauer et al., Proc Natl Acad Sci USA 1986, 83:3746-3750). FIG. 6B is a graph depicting the number of mismatches between microRNAs plotted against the number of known mouse microRNAs (Rfam 3.0, January 2004). Each microRNA was aligned pairwise to every other microRNA and was assigned to the group (No. mismatches) corresponding to the least number of mismatches that it had to another microRNA.
FIG. 7A shows the profile of microRNA expression in the developing mouse brain. Relative expression levels are shown for the 66 microRNAs that changed significantly (ANOVA, P<0.001) (i.e., more than two-fold) are shown in five columns corresponding to the five developmental time points. The gray scale at the bottom indicates relative signal intensities. The microRNA expression profile was sorted using a hierarchical clustering method, and major clusters are shown ordered according to the time that expression peaks. Gene names and a quantitative description of microRNA expression levels are presented in Table 2. FIG. 7B shows developmental time points grouped using the same hierarchical clustering method and gene set as shown in FIG. 7A.
FIG. 8A shows the profile of microRNA expression at embryonic day 12.5 in mouse brain for those microRNAs that exhibit a sharp peak in expression at this stage. The oligonucleotide sequences EAM261 (SEQ ID NO:360), EAM206 (SEQ ID NO:361), EAM119 (SEQ ID NO:362), EAM279 (SEQ ID NO:363), EAM268 (SEQ ID NO:364), EAM137 (SEQ ID NO:365), EAM105 (SEQ ID NO:366), EAM289 (SEQ ID NO:367), EAM255 (SEQ ID NO:368), EAM182 (SEQ ID NO:369), EAM195 (SEQ ID NO:370), and EAM 194 (SEQ ID NO:371) are also shown. Similar sequences are indicated with brackets. FIG. 8B shows the profile of microRNA expression in adult rat brain for microRNAs that exhibit a single sharp peak in expression at the adult stage. The oligonucleotide sequences EAM235 (SEQ ID NO:372), EAM282 (SEQ ID NO:373), EAM 159 (SEQ ID NO:374), EAM225 (SEQ ID NO:375), EAM237 (SEQ ID NO:376), EAM226 (SEQ ID NO:377), EAM227 (SEQ ID NO:378), EAM228 (SEQ ID NO:379), EAM 156 (SEQ ID NO:380), EAM276 (SEQ ID NO:381), EAM133 (SEQ ID NO:382), EAM175 (SEQ ID NO:383), EAM216 (SEQ ID NO:384), and EAM202(SEQ ID NO:385) are also shown. Methods used in assembling this profile are the same as those described above for FIGS. 7A and 7B. Brackets indicate closely related sequences.
FIG. 9 shows a side-by-side comparison of microarray hybridization signal data (right panel) with representative developmental northern blots of microRNAs (left panel). Northern blots were prepared and microarray analysis was done using the same starting material. Y-axis for the microarray data refers to the averaged mean signal intensities (×10−3), and error bars are standard errors of the mean. Since northern blots were exposed for different lengths of time, the intensities of the signals on northern blots cannot be directly compared to those from the microarrays. A probe against U6 snRNA was hybridized to the same blots as a control to ensure that the wells were evenly loaded.
FIG. 10 is a schematic diagram illustrating the methods used for microarray design and data analysis.
FIG. 11 shows an exemplary microarray scan.
FIG. 12 shows the relative expression level of each of the indicated microRNAs at the indicated developmental stages.
FIG. 13 shows three scatter plots depicting the correlations of four hybridizations at time point E12.5. For each graph, the axes show averaged mean spot intensities for all probes from a given data set, as indicated.
FIG. 14 shows four scatter plots that depict the correlations of the hybridization data obtained at E12.5 with the data from the other indicated timepoints (each set of data is averaged over four hybridizations).



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods and compositions for the analysis of microRNA expression.
Such methods and compositions are useful for the analysis of microRNAs in virtually any biological sample. In general, the microRNA microarray system that we have developed can be used to determine the expression of all known microRNAs simultaneously for virtually any set of experimental conditions or constraints. Such methods are particularly useful for the analysis of micro RNAs that function in human health and disease and for the identification of drugs that modulate microRNA function.
As described below, we determined the temporal expression pattern of 138 microRNAs in the developing mouse brain and found that the levels of 66 microRNAs changed significantly during development. We identified sets of genes with similar expression patterns, including genes that peaked in expression at different stages of development using methods and compositions that we developed for microRNA expression analysis.
Identification of microRNAs from Developing Rat and Monkey Brains
To analyze microRNAs expressed in the developing mammalian brain, we cloned small 18-26 nucleotide RNAs from the neocortex and hippocampus of a 12-day postnatal rat (Rattus norvegicus) and from the cerebral wall of a 114-day old rhesus fetal monkey (Macaca mulatta) (Table 1).








TABLE 1







Rattus norvegicus microRNAs
Macaca mulatta microRNAs












Name
No. times cloned
Size Range
Name
No. times cloned
Size Range















rno-miR-b
2






rno-let-7a
3
22
mml-let-7a
15
21





mml-let-7a or c
1
18


rno-let-7b
1
23
mml-let-7b
20
22-23


rno-let-7c
10
22
mml-let-7c
9
21-22


rno-let-7d
1
22
mml-let-7d
1
22





mml-let-7e
3
20-22





mml-let-7f
3
22





mml-let-7g
2
22


rno-let-7i
1
22
mml-let-7i
2
22


rno-miR-7
5





mml-miR-7-1
1
22


rno-miR-9
2

mml-miR-9
9
21-23


rno-miR-16
2
22
mml-miR-16
2
22


rno-miR-17-5p
3
23
mml-miR-17-5p
2
22-23


rno-miR-24
6
21-22





mml-miR-26a
3
21-22


rno-miR-26b
1
22


rno-miR-28
1
22


rno-miR-29a
4
22


rno-miR-29b
7
22-23


rno-miR-29c
2
20, 22


rno-miR-30b
1
22
mml-miR-30b
2
22


rno-miR-30c
3
23-24
mml-miR-30c
1
21





mml-miR-33
2
20


rno-miR-92
2
22
mml-miR-92


rno-miR-93
1
23


rno-miR-99a
1
21
mml-miR-99a
4
20-22


rno-miR-99b
2
21, 22
mml-miR-99b
2
22





mml-mir-100
1
22


rno-miR-101b
1
22


rno-miR-103
3
23
mml-miR-103
2
22-23





mml-miR-103 or 107
1
21


rno-miR-124a
19
19-22
mml-miR-124a
97
18-23


rno-miR-125a
2
22, 24
mml-miR-125a
4
22-23


rno-miR-125b
12
21-22
mml-miR-125b
17
20-22





mml-miR-126
1
21





mml-miR-126*
1
22


rno-miR-127
1
20


rno-miR-128a
3
21-22
mml-miR-128a
9
22


rno-miR-128a or b
2
21
mml-miR-128a or b
17
18-21


rno-miR-128b
1
21
mml-miR-128b
8
22


rno-miR-129
2
21-22
mml-miR-129-2
1
22


rno-miR-130a
1
22


rno-miR-132
6
22


rno-miR-136
2
23
mml-miR-136
1
23





mml-miR-137
1
23


rno-miR-138
5
23-24


rno-miR-139
1
23





mml-miR-140
1
22


rno-miR-140*
1
22


rno-miR-142-3p
1
23


rno-miR-145
1
23
mml-miR-145
2
22


rno-miR-146
2
23





mml-miR-149
2
23


rno-miR-150
4
22-23


rno-miR-154
1
22





mml-miR-181a or 213
4
20-25





mml-miR-181b
1
24





mml-miR-181c
1
21


rno-miR-185
2
22-23
mml-miR-185
1
23


rno-miR-191
3
23-24





mml-miR-195
1
22


rno-miR-213
1
22





mml-miR-221
3
22-23


rno-miR-300
1
21


rno-miR-323
1
22


rno-miR-324
4
23


rno-miR-325
1
22


rno-miR-338
5
23


rno-miR-342
1
25


rno-miR-345
1
22





152

Total
261





Legend:


The rat (rno) and monkey (mml) microRNA names are indicated. Two microRNA names are assigned to the same clone when the cloned sequence is too short to distinguish between the microRNAs. mml-miR-7 and mml-miR-129 are encoded by three and two distinct genomic loci, respectively, although the sequences immediately adjacent to these microRNA sequences differ. The sequences we cloned for mml-miR-7-1 and mml-miR-129-2 were one base longer than that shared by the microRNAs, allowing us to determine the loci from where they originated, as indicated by −1 and −2. Notation follows the Rfam repository guidelines (Griffiths-Jones et al., Nucleic Acids Res 31:439-441, 2003)






Table 1 lists the identity, frequency, and size range of microRNAs cloned from the cortex and hippocampus of 12-day postnatal R. norvegicus and the cortex of a 114-day old M. mulatta fetus. In both species, at these stages, most neurons have been generated and have begun synaptogenesis (Rakic, Science 241:170-176, 1988; Angevine Nature 192:766-768, 1961). We identified a total of 1451 sequences, four hundred thirteen of which corresponded to microRNA sequences. These four hundred thirteen sequences defined sixty-eight unique microRNAs that are listed in Table 1. These sequences generate stem-loop precursors and have corresponding orthologous sequences in the rat and/or human genomes. In all but one case, the microRNAs identified corresponded to known microRNAs from other species.
Rat microRNA Precursors
Using the assembly of the rat genome, we identified candidate genomic locations for all of our rat microRNAs. All of these microRNAs have orthologs in mouse, but none of these microRNAs were previously identified in the rat. An alignment of the top BLAST hit of each mouse microRNA precursor sequence (Rfam, Release 3.0) against the rat genome sequence (public release draft genome assembly, version 3.1) is shown in FIG. 1A. In addition, predicted precursor secondary structures are shown (FIG. 1B) for each rat microRNA gene for which the precursor sequence differs from that of the corresponding mouse precursor. We used the mfold algorithm to make secondary structure predictions (Zuker Nucleic Acids Res, 31:3406-3415, 2003).
One of these microRNAs is novel. It differs in sequence from any microRNA previously described and is conserved in the mouse and human genomes. We named this new microRNA rno-miR-B (FIG. 2A). FIG. 2B shows an alignment of the predicted precursor sequences from human and mouse with the novel microRNA miR-B, which we identified from rat. The cloned sequence, which corresponds to the mature microRNA, is shown in bold (FIG. 2B). The single mismatch is indicated by an asterix (*). The accession number for each sequence is given. The predicted secondary structures for the corresponding genes from mouse and human are shown in FIG. 3. The human and mouse genomic sequences for candidate miR-B precursors are identical. The sequence of the mature microRNA is in gray. The residue in the rat genomic sequence that is different from the mouse and human genomic sequences is indicated with an arrow. We used the mfold algorithm to make secondary structure predictions (Zuker Nucleic Acids Res, 31:3406-3415, 2003). We have not been able to detect miR-B in mouse brain using northern blots.
In addition to microRNAs, we cloned 13 small RNAs that do not satisfy all criteria to be considered microRNAs. While twelve of these small RNAs are not predicted to form stem-loop structures, smallRNA-1 from monkey has a predicted stem-loop precursor sequence that is characteristic of microRNAs. In smallRNA-1 the stem-loop ends on the final base of the microRNA. Given this atypical structure, we classify this RNA as a small non-coding RNA, rather than a microRNA. A northern blot for smallRNA-1 revealed a high molecular weight band that may represent a precursor RNA. While there is no perfect match to smallRNA-1 in the human genome sequence released to date, a presumptive precursor based on the mouse genomic sequence is shown in FIG. 4. The cloned sequence is in gray. The other small RNAs are not predicted to form stem-loop structures.
Of the fifty-two rat microRNA sequences cloned, twenty-seven were previously cloned from rat primary cortical neurons (Kim et al., Proc Natl Acad Sci USA 101:360-365, 2004) (FIG. 1B). For 21 of the 52 microRNAs from rat and 14 of the 40 microRNAs from monkey, we isolated only a single clone, indicating that our surveys are not saturated.
microRNA miR-124a was isolated nineteen times from rat and ninety-seven times from monkey. Mouse miR-124a, miR-128, miR-101, and miR-132 were previously reported to be expressed specifically in brain (Lagos-Quintana et al., Curr Biol 12:735-739, 2002). Using Northern blot analysis, we found that rat miR-138 is also expressed exclusively in brain (FIG. 5). The probe was identical to EAM125. Total RNA isolated from various adult rat tissues (AMBION, Austin, Tex.) was size-separated on a denaturing PAGE gel that was loaded with 12 μg RNA per lane. After separation the RNA was transferred to a nylon membrane and used for hybridization. Equal loading was verified using a probe for U6 snRNA.
microRNA Microarrays for the Study of Temporal and Spatial Patterns of microRNA Expression
Prior analyses of microRNA expression relied on the individual characterization of each microRNA using dot blots, northern blots, and cloning strategies (Lim et al., Science, 299:1540, 2003; Kim et al., Proc Natl Acad Sci USA 101:360-365, 2004; Lee et al., Science 294:862-864, 2001; Lagos-Quintana Science 294:853-858, 2001; Lau et al., Science 294:858-862, 2001; Dostie RNA 9:180-186, 2003; Krichevsky RNA 9:1274-1281, 2003; Sempere Genome Biol 5:R13, 2004). We now describe a highly scalable approach using a microarray that provides for the analysis of microRNA expression patterns for a large number of samples simultaneously. We arrayed 138 oligonucleotides complementary to microRNAs (probes) corresponding to the 68 mammalian microRNAs we isolated from rat and monkey brains and to 70 mammalian microRNAs isolated by others from a variety of mouse tissues and mammalian cell lines as well as to predicted microRNAs. In addition, we included a set of control probes as well as 19 probes corresponding to presumptive small RNAs that we and others identified, but that do not satisfy all criteria for a microRNA.
Each probe contained a free amine group at the 5′ terminus and was printed onto an amine-binding glass slides. The probes were covalently linked to the glass surface. All probes were printed in quadruplicate.
microRNA Labeling Method
We developed a method for preparing microRNA samples for microarray analysis. Several methods for mRNA sample labeling for microarray analysis have been described (Duggan et al., Nat Genet 21(1 Suppl):10-14, 1999; Schena Science 270:467-470, 1995; Nimmakayalu et al., Biotechniques 28:518-522, 2000; Gupta et al., Nucleic Acids Res 31:e13, 2003), but none of these methods is suitable for labeling RNAs as small as microRNAs. To fluorescently label small RNAs we adapted strategies for RNA ligation and reverse transcriptase (RT-) PCR that were devised for microRNA cloning (Lee et al., Science 294:862-864, 2001; Lagos-Quintana et al., Science 294:853-858, 2001; Lau et al., Science 294:858-862, 2001). Briefly, 18-26 nucleotide RNAs were size-selected from total RNA using denaturing polyacrylamide gel electrophoresis. Oligonucleotide linkers were attached to the 5′ and 3′ ends of the small RNAs and the resulting ligation products were used as templates for an RT-PCR reaction with 10 cycles of amplification. The sense-strand PCR primer had a Cy3 fluorophore attached to its 5′ end, thereby fluorescently labeling the sense strand of the PCR product. The PCR product was denatured and then hybridized to the microarray. As in microarray analysis, a labeled sample used for hybridization is referred to as the target. While significant biases in amplification are a problem when heterogeneously sized mRNAs are amplified, such biases are less likely to be problematic when amplifying microRNAs given their short uniform lengths. microRNA cloning frequencies that were obtained using a similar amplification strategy correlated well with expression levels as assayed by quantitative northern blots (Lim et al., Genes Dev 2003, 17(8):991-1008). Since RNA is amplified prior to hybridization, relatively low amounts of starting material may be used with this method (Lim et al., Science, 299:1540, 2003; Kim et al., Proc Natl Acad Sci USA 101:360-365, 2004; Lee et al., Science 294:862-864, 2001; Lagos-Quintana Science 294:853-858, 2001; Lau et al., Science 294:858-862, 2001; Dostie RNA 9:180-186, 2003; Krichevsky RNA 9:1274-1281, 2003; Sempere Genome Biol 5:R13, 2004).
microRNA Hybridization
We optimized the conditions for hybridization to our microarray as follows. Because of the small size of microRNAs, it is difficult to design oligonucleotides (array probes), where the probe and the target melting temperatures are the same. Differences between the melting temperatures of the probe and the target were expected to result in non-specific binding between the probe and the target when hybridizations are performed at low temperatures, which-allows for the detection of bound probe/target pairs with low melting temperatures. If hybridizations are performed at high temperatures to detect probe/target pairs with high melting temperatures, we expected to find less nonspecific binding, but to find a decrease in sensitivity.
We tested a range of hybridization temperatures, and, based on the signal of microRNA probes versus control probes, we determined that a hybridization temperature of 50° C. was a reasonable compromise between sensitivity and specificity. Even at 50° C., specificity as assayed by comparing microarray spot signal intensities from matched and mismatched probes varied among the microRNAs assayed. As expected, specificity at 50° C. was negatively correlated with calculated melting temperatures (FIG. 2A).
Control probes with two internal mismatches on the microarray were included for a subset of the microRNA probes as described in more detail below. In all cases the cumulative signal from 10 hybridizations for the mismatched probe was equal to or lower than that for the microRNA probe, but differences in the ratio of the matched to mismatched probe signal ranged widely (FIG. 6A). Given these data, we did not expect that the microRNA microarray would reliably distinguish between microRNAs that have only one or a few mismatches. Surprisingly, this did not present a problem, most likely because the most closely related paralogs for identified microRNAs differ by five mismatches or more (FIG. 6B). Thus, the signal from a mismatched control probe is typically caused by cross-hybridization with the microRNA for which it was designed. Given these results, it is possible that differences in the apparent expression of a given microRNA may be altered by the expression of microRNA paralogs (FIG. 2A). Control probes corresponding to unrelated mRNA subsequences or synthetic probes that do not correspond to known microRNAs did not show signals above background.
Analysis of microRNA Expression During Mouse Brain Development
We isolated small RNAs from mice at five developmental stages, embryonic days 12.5 and 17.5 (E12.5 and E17.5), postnatal days 4 and 18 (P4 and P18), and 4-month old adults. E12.5-E17.5 spans a period of major neuronal proliferation and migration in the mouse brain, in particular the birth and subsequent migration of most neurons in the ventricular zone epithelium (Chenn et al., Molecular and Cellular Approaches to Neural Development. Edited by Cowan W M, Jessell T M, Zipursky S L. New York: Oxford University Press; 1997: 440-473). Between postnatal days P4 and P18 major sensory inputs are established. For example, eye opening occurs around P13 and is thought to result in activity-dependent neuronal remodeling (Chen et al., Neuron 28:955-966, 2000).
We purified and size-selected RNA from whole mouse brains. For each sample, the products of four independent RNA amplifications based on two independent RNA ligations were hybridized to the array. A detailed description of our analysis of the microarray data is presented below. Of the 138 microRNAs and 19 small RNAs represented by the probe set, 116 (74%) were expressed robustly (greater than 75-fold over the level of background controls) during at least one timepoint. Of these, eighty-three (71%) changed significantly during the period surveyed (analysis of variance, ANOVA, P<0.001) and sixty-six (57%) changed more than two-fold.
We grouped microRNAs that changed more than two-fold in expression during the period analyzed using a hierarchical clustering algorithm (FIGS. 7A and 8A) (Eisen Proc Natl Acad Sci USA 95:14863-14868, 1998). Hierarchical clustering methods organize data in a tree structure, based on similarity (FIG. 7B). A group of microRNAs peaked at each of the developmental timepoints. The signal from thirty-four of the sixty-six probes that changed more than two-fold peaked in the fetus (E12.5 and E17.5), suggested that these microRNAs function in early development (FIG. 4A). Nine microRNAs peaked in expression level during the neonate (P4) stage, while eleven other microRNAs peaked at the juvenile (P18) stage. Twelve microRNAs were expressed at their highest level at the adult stage (FIG. 8B). These data indicate that murine brain development involves a wave of expression of sequential classes of microRNAs (FIG. 7A).
We also grouped the developmental timepoints according to their microRNA expression pattern using hierarchical clustering. We found that samples from stages that are developmentally proximal had the most similar microRNA expression patterns (FIG. 7B), indicating that a microRNA expression profile can be a marker of developmental stage. Examination of temporal clusters revealed that probes with similar sequences showed correlated expression, as exemplified by miR-181a, miR-181b, miR-181 c, smallRNA-12 as shown in FIG. 8A and further exemplified by miR-29a, miR-29b and miR-29c as shown in FIG. 8B, respectively. We found that four (smallRNA-2 5′-TGGTGTCAGAAGTGGGATAC-3′(SEQ ID NO:1); smallRNA-12 5′-ACTCACCGAGAGCGTTGAATGTT-3′(SEQ ID NO:2); let-7h 5′-AACTGTACACACTACTACCTCA-3′(SEQ ID NO:3); miR-33b 5′-CAATGCAACAGCAATGCAC-3′(SEQ ID NO:4)) of the sixty-six RNAs that changed more than two-fold were small RNAs rather than microRNAs. The temporal regulation of these small RNAs indicated that they are likely to function in development.
Validation of Microarray Results Using Northern Blots
To validate our microarry results, we performed northern blots of eight microRNAs that were robustly expressed during at least at one point during development according to our microarray data (FIG. 9). The relative change in microRNA expression as determined using microarray analysis and northern blot analysis was consistent (FIG. 9). For example, for both microarray and northern blot analysis indicated that miR-29b was almost undetectable at the embryonic and P4 stages; expression became detectable at P18, and the microRNA was strongly expressed in the adult. In only a few cases did there seem to be discrepancies between microarray and northern blot analysis; for example, there were small differences in the relative levels of expression of miR-138 at P4 and adult stages differed between northern blot and microarray analysis. Microarrays offer a high-throughput method that provides for the analysis of microRNA expression patterns.
The development of microarray technology for profiling the expression of microRNAs and other small RNAs is described herein and a working example is provided that shows the results of applying this technology to the developing mammalian brain. Recently, Krichevsky et al. described the temporal expression of 44 microRNAs during mouse brain development (Krichevsky et al., RNA, 9:1274-1281, 2003). The Krichevsky study used a dot-blot array approach and required the direct radioactive labeling of individual microRNAs. In contrast, our approach used a glass microarray and RT-PCR/fluorescent labeling. Despite differences in sample selection as well as in the number of microRNAs analyzed, where the two studies overlap, the data analyses typically agree. Thus, our strategy, which is demonstrably no less sensitive than that of Krichevsky, offers two significant advantages that Krichevsky's techniques does not; it is highly scalable and allows for the high-throughput analysis of even small biological samples.
As described herein, microRNA microarrays offer a new tool that provides for the analysis of microRNAs. It is likely that many of the developmentally regulated microRNAs described herein function in the control of mammalian brain development, possibly by controlling developmental timing, analogous to the roles of the lin-4 and let-7 microRNAs in C. elegans. 
The results described above were carried out using the following methods.
microRNA Cloning
We isolated RNAs and cloned microRNAs from R. norvegicus and M. mulatta using methods described previously (Lagos-Quintana et al., Science 294:853-858, 2001) except that the samples were not dephosphorylated during the cloning procedure.
Microarray Printing and Hybridization
Microarray probes were oligonucleotides (identified herein as EAM followed by a number) having sequences that are complementary to microRNAs. Each probe was modified with a free amino group linked to its 5′ terminus through a 6-carbon spacer (IDT) and was printed onto an amine-binding slide (CodeLink, Amersham Biosciences Little Chalfont, UK). Control probes contained two internal mismatches resulting in either C-to-G or T-to-A changes. The sequences of the control and microarray probes (SEQ ID NOS:5-219) are shown in Table 2, which also provides a summary of the microarray data. The sequences of Table 2, along with their names and sequence identifiers, are shown in Table 3.


























Oligo

Small RNA

Oligo
Tm

E12.5

E17.5

P4

P18

Adult



ID
Oligo sequence
(Rfam 3.0)
A B C
length
(NN)
E12.5
SEM
E17.5
SEM
P4
SEM
P18
SEM
Adult
SEM































EAM101
TCCATCATCAAAACAAATGGAGT
mmu-miR-136
−−−
23
67.9
2,566.6
553.8
3.099.4
492.7
5,478.9
486.3
5,422.4
438.2
2,195.1
271.8



 


EAM102
TCCATCATGAAAAGAAATGGAGT
control
−−−
23
67.3
208.0
21.8
306.0
19.7
495.5
31.6
457.3
82.9
213.9
45.4


 


EAM103
TGGCATTCACCGCGTGCCTTA
mmu-miR-124a
+++
21
78.1
10,479.0
554.2
 7,301.8
350.5
7,185.7
296.3
7,921.3
595.8
8,544.7
716.9


 


EAM104
TGGCATTCAGCGGGTGCCTTA
control
−−−
21
77.2
6,552.3
677.5
 4,992.1
223.8
5,475.9
272.2
5,481.6
467.6
5,419.0
332.1


 


EAM105
TCACAAGTTAGGGTCTCAGGGA
mmu-miR-125b
+++
22
67.8
4,367.2
445.3
 6,279.3
280.8
4,884.0
464.5
3,123.8
274.0
3,238.3
399.4


 


EAM106
TCACAAGTAAGGGTGTCAGGGA
control
−−−
22
68.5
2,203.5
163.3
 3,310.7
134.6
2,250.2
111.4
1,400.7
145.9
1,456.1
141.5


 


EAM107
TGTTCCTGCTGAACTGAGCCA
mmu-miR-24
−−−
21
70.4
748.7
104.7
715.9
87.5
550.8
92.7
1,061.9
220.2
1,887.1
239.1


 


EAM108
TGTTCCTGGTGAAGTGAGCCA
control
−−−
21
70.1
546.4
57.9
528.4
71.1
556.9
54.4
869.5
107.4
1,345.8
140.0


 


EAM109
AACAACAAAATCACTAGTCTTCCA
mmu-miR-7
+++
24
64.9
1,917.8
181.4
 1,929.6
162.9
1,020.1
131.5
837.8
128.0
666.0
80.7


 


EAM110
AACAACAAAATGAGTAGTCTTCCA
control
−−−
24
64.9
793.5
86.8
701.8
105.0
489.1
49.7
330.4
45.9
266.5
31.4


 


EAM1100
GCATGCATGCATGCATGCATG
control
−−−
21
76.1
−0.5
0.1
0.0
0.1
−0.3
0.1
−0.1
0.1
0.3
0.2


 


EAM1101
GTGGTAGCGCAGTGCGTAGAA
control
−−−
21
70.5
0.3
0.1
0.4
0.1
0.1
0.1
0.3
0.1
1.2
0.4


 


EAM1102
GGTGATGCCCTGAATGTTGTC
control
−−−
21
68.9
0.2
0.2
0.3
0.1
0.3
0.1
0.2
0.1
0.2
0.1


 


EAM1103
TGTCATGGATGACCTTGGCCA
control
−−−
21
73.0
0.3
0.1
0.5
0.1
0.1
0.1
−0.8
0.2
−0.5
0.1


 


EAM1104
CTTTTGACATTGAAGGGAGCT
control
−−−
21
65.5
1.0
0.4
0.4
0.1
0.4
0.1
0.3
0.1
0.3
0.0


 


EAM111
TAACTGTACAAACTACTACCTCA
mmu-let-7g
+++
23
56.4
1,585.3
124.9
 2,631.4
163.8
2,519.8
324.0
2,208.7
222.5
2,944.3
244.0


 


EAM112
TAACTGTAGAAAGTACTACCTCA
control
−−−
23
55.9
18.6
3.4
42.2
8.5
37.5
7.7
31.6
8.4
49.2
7.8


 


EAM113
ACAGGTTAAAGGGTCTCAGGGA
mmu-miR-125a
−−−
22
68.7
1,916.1
253.8
 2,181.2
307.8
2,051.6
453.0
1,394.4
263.3
2,751.5
445.9


 


EAM114
ACAGGTAAAAGGGTGTCAGGGA
control
−−−
22
69.3
852.4
94.9
965.0
99.9
592.0
65.6
609.5
61.3
1,396.0
109.5


 


EAM115
CGCCAATATTTACGTGCTGCTA
mmu-miR-16
+++
22
70.2
1,273.8
134.0
 1,571.9
300.7
1,398.5
286.0
910.3
186.0
995.4
149.3


 


EAM116
CGCCAATATTAAGGTGCTGCTA
control
−−−
22
69.5
872.5
115.7
 1,009.6
136.8
696.3
80.6
416.9
48.0
281.0
27.9


 


EAM117
TAACCGATTTCAAATGGTGCTA
mmu-miR-29c
−−−
22
67.2
154.4
40.6
292.8
60.6
949.9
312.6
3,313.3
290.1
5,406.9
146.4


 


EAM118
TAACCGATTTGAAAAGGTGCTA
control
−−−
22
66.7
4.8
0.9
4.5
0.6
2.2
0.7
28.5
8.8
103.3
31.9


 


EAM119
AACACTGATTTCAAATGGTGCTA
mmu-miR-29b
+++
23
66.2
115.9
16.9
204.1
29.5
227.1
35.0
2,617.0
203.4
5,125.9
373.6


 


EAM120
AACACTGATTTGAAAAGGTGCTA
control
−−−
23
65.7
18.3
3.8
22.5
3.9
80.4
10.3
655.5
80.4
1,225.8
77.3


 


EAM121
CACAAGATCGGATCTACGGGT
mmu-miR-99a
+++
21
68.0
3,074.1
472.8
4,504.2
497.3
2,168.9
238.9
1,207.4
131.0
607.1
88.1


 


EAM122
CACAAGATGGGATGTACGGGT
control
−−−
21
68.5
775.9
51.0
1,115.3
76.8
720.3
37.6
429.6
32.6
240.1
34.5


 


EAM123
AACTATGCAACCTACTACCTCT
mmu-let-7d
−−−
22
59.4
4,723.1
330.2
4,694.2
353.2
4,971.2
500.8
5,081.4
529.3
6,340.0
327.6


 


EAM124
AACTATGCAACGTAGTACCTCT
control
−−−
22
60.1
500.3
82.7
1,040.7
93.3
1,438.5
116.9
1,531.2
131.4
1,650.9
153.2


 


EAM125
CGGCCTGATTCACAACACCAGCT
mmu-miR-138
−−−
23
77.2
643.0
143.6
1,170.0
201.3
3,454.0
688.3
4,646.7
785.8
3,313.7
477.1


 


EAM126
CGGCCTGATTGAGAACACCAGCT
control
−−−
23
76.6
448.2
83.9
808.9
115.4
1,733.3
254.2
2,227.5
289.2
1,450.0
182.6


 


EAM127
TCATAGCCCTGTACAATGCTGCT
mmu-miR-103
−−−
23
70.8
1,163.8
181.0
1,531.3
265.1
1,663.4
442.6
1,492.5
337.1
932.0
188.7


 


EAM128
TCATAGCCCTGAAGAATGCTGCT
control
−−−
23
72.3
904.3
155.8
875.6
126.5
651.2
88.5
900.0
119.2
448.3
84.5


 


EAM129
AGGCATTCACCGCGTGCCTTAT
mmu-miR-124a
−−−
22
76.8
11,594.7
1,000.9
8,352.1
590.8
10,020.2
561.9
9,323.5
715.1
10,618.3
1,171.4


 


EAM130
AGGCATTCAGCGGGTGCCTTAT
control
−−−
22
76.0
7,004.6
938.7
5,699.5
450.9
6,117.8
553.5
5,927.1
421.3
8,189.1
788.6


 


EAM131
ACAGGCCGGGACAAGTGCAATAT
mmu-miR-92
+++
23
75.9
2,733.4
317.5
893.1
110.5
255.5
49.9
126.8
21.9
142.2
26.1


 


EAM132
ACAGGCCGGGAGAAGAGCAATAT
control
−−−
23
74.6
800.4
64.5
186.9
39.4
72.9
11.1
37.8
7.9
54.3
6.4


 


EAM133
ACACCAATGCCCTAGGGGATGCG
mmu-miR-324-5p
+++
23
80.0
286.4
40.8
222.0
31.5
161.7
27.1
114.4
20.6
101.7
17.3


 


EAM134
ACACCAATGGCGTAGGGGATGCG
control
−−−
23
80.8
177.7
21.4
187.6
26.9
167.9
34.5
168.6
28.8
93.2
15.0


 


EAM135
CAACAAACATTTAATGAGGCC
mmu-miR-B
+++
21
64.4
−0.6
0.2
−1.8
0.4
−1.4
0.2
2.1
1.7
3.4
1.4


 


EAM136
CAACAAAGATTAAATGAGGCC
control
−−−
21
63.8
3.5
0.4
2.6
0.3
3.3
0.5
2.9
0.5
5.2
0.6


 


EAM137
CCGACCATGGCTGTAGACTGTTA
mmu-miR-132
+++
23
70.9
16.6
5.0
54.7
14.7
102.3
32.7
280.1
59.4
808.3
146.9


 


EAM138
CCGACCATGGGTGAAGACTGTTA
control
−−−
23
72.8
14.0
5.0
43.1
10.1
31.8
9.7
193.8
50.2
438.2
78.8


 


EAM139
TAACCCATGGAATTCAGTTCTCA
mmu-miR-146
+++
23
68.1
419.7
88.1
662.1
145.6
1,703.2
518.6
1,612.3
341.4
669.9
162.1


 


EAM140
TAACCCATGGAAATGAGTTCTCA
control
−−−
23
68.1
1.3
0.3
0.9
0.1
0.8
0.1
3.1
0.9
1.3
0.4


 


EAM141
TAACTATACAATCTACTACCTCA
mmu-let-7f
−−−
23
53.6
3,685.7
347.4
3,918.5
372.9
4,705.6
333.1
3,671.1
176.2
4,305.7
239.8


 


EAM142
TAACTATACAATGTAGTACCTCA
control
−−−
23
54.2
1.7
1.5
4.8
2.3
7.1
3.4
8.4
4.3
12.1
4.6


 


EAM143
TAACCATACAACCTATTACCTCA
smallRNA-9
+−−
23
61.2
4,624.5
272.5
6,065.4
573.7
5,549.0
508.8
5,381.2
456.6
5,382.2
351.1


 


EAM144
TAACCATAGAACGTATTACCTCA
control
−−−
23
61.4
1.1
0.2
2.1
0.5
1.9
0.5
1.3
0.4
1.5
0.3


 


EAM145
AACCATACAACCTACTACCTCA
mmu-let-7c
+++
22
60.3
7,443.7
624.0
7,035.7
1,002.3
7,055.3
882.9
7,347.4
925.7
8,854.7
641.2


 


EAM146
AACCATACAAGCTAGTACCTCA
control
−−−
22
60.7
2,914.2
332.1
3,841.4
333.9
4,215.1
458.8
3,283.2
258.1
3,696.1
171.0


 


EAM147
AACCACACAACCTACTACCTCA
mmu-let-7b
+++
22
63.1
6,198.0
466.8
7,186.7
856.7
5,764.8
594.8
7,353.0
1,059.8
7,098.9
417.2


 


EAM148
AACCACACAAGCTAGTACCTCA
control
−−−
22
63.5
2,334.0
238.0
3,909.2
307.2
4,671.7
587.2
4,016.0
371.7
4,336.3
208.3


 


EAM149II
GCATTCACCCGCGTGCCTTA
mir-124b#
+−−
20
75.4
11,150.9
1,375.8
6,018.1
1,009.9
7,196.2
1,151.7
6,949.5
1,326.9
7,285.2
1,404.3


 


EAM150II
TCATACAGCTAGATAACCAAAGA
mmu-miR-9
−−−
23
61.4
4,877.7
482.6
6,556.2
983.2
5,796.1
459.3
2,967.1
341.2
2,211.1
354.7


 


EAM151
ACAAGATCGGATCTACGG
mmu-miR-99a
−−−
18
58.6
1,886.8
349.5
2,625.7
292.1
2,289.9
380.0
1,520.5
294.5
313.5
41.1


 


EAM152
ACTTTCGGTTATCTAGCTTTAT
mmu-miR-9*
+++
22
59.7
687.9
146.6
651.2
110.6
1,279.9
168.1
300.0
45.9
214.3
40.2


 


EAM153
AACTATACAACCTACTACCTCA
mmu-let-7a
+++
22
55.2
8,098.5
741.1
6,883.1
447.6
7,513.1
446.0
7,661.8
535.1
8,165.0
730.7


 


EAM154
AAAGAGACCGGTTCACTGTGA
mmu-miR-128b
−−−
21
66.0
2,017.8
395.2
5,627.3
631.1
6,962.5
253.6
6,732.5
523.4
7,150.9
570.7


 


EAM155
TCCATCATCAAAACAAATGGAGT
mmu-miR-136
+++
23
67.9
2,428.6
268.3
3,319.0
191.5
3,849.1
262.1
4,409.4
231.9
2,022.0
205.2


 


EAM156
ACTCACCGAGAGCGTTGAATGTT
smallRNA-12
+−−
23
71.9
4,086.4
267.5
3,199.8
243.6
2,266.9
110.5
1,714.3
126.9
1,748.9
225.4


 


EAM157
TGGTGTCAGAAGTGGGATAC
smallRNA-2
+−−
20
61.3
246.6
46.6
183.7
30.4
82.1
13.5
61.2
12.8
35.8
6.5


 


EAM158
TACAGCTAAATAACCAAAGA
smallRNA-13
+−−
20
54.9
290.2
78.7
341.3
112.2
228.8
59.6
98.3
39.6
114.7
34.1


 


EAM159
ATGCCCTTTTAACATTGCACTG
mmu-miR-130a
+++
22
68.9
2,204.4
177.6
1,392.2
154.2
877.8
111.2
571.8
76.9
209.0
36.6


 


EAM160
AACCTATCCTGAATTACTTGAA
mmu-miR-26b
+++
22
60.1
1,452.0
212.1
2,227.8
311.0
2,037.4
180.4
2,099.7
139.0
1,033.1
105.2


 


EAM161
CTCAATAGACTGTGAGCTCCTT
mmu-miR-28
+++
22
62.3
109.2
14.8
115.9
19.6
144.8
30.8
128.3
21.4
142.6
18.4


 


EAM162
ATCAAGGTCCGCTGTGAACACG
[mmu-miR-
+++
22
73.7
116.5
25.9
112.0
22.1
66.1
12.6
53.9
16.7
46.2
12.4




124a-as]


 


EAM163
TCCATAAAGTAGGAAACACTACA
mmu-miR-142-3p
+++
23
61.1
196.0
39.4
288.9
40.4
249.1
58.3
197.8
28.6
49.1
7.5


 


EAM164
GAGTGCTTGCTAGGTGCCAAG
smallRNA-3
+−−
21
69.2
1.6
0.7
0.1
0.4
0.9
0.7
−0.7
0.4
−0.2
0.1


 


EAM165
GGGAGTGAAGACACGGAGCCAGA
mmu-miR-149
−−−
23
76.4
489.7
76.4
387.2
85.9
386.8
80.9
222.0
49.6
115.1
24.8


 


EAM166
GCCTATCCTGGATTACTTGAA
mmu-miR-26a
−−−
21
63.2
1,215.2
188.9
1,977.5
200.7
2,120.3
174.4
1,818.8
215.7
1,896.3
186.9


 


EAM167
GTTGTGGTCACTTACAATT
smallRNA-4
+−−
19
53.1
0.2
0.1
0.2
0.1
0.3
0.1
0.2
0.1
0.5
0.1


 


EAM168
CTATACAACCTCCTACCTCA
mmu-let-7e
+++
20
55.6
3,703.9
535.7
4,698.0
249.3
4,131.1
249.1
3,888.9
225.2
3,182.2
262.8


 


EAM169
AACAGCACAAACTACTACCTCA
mmu-let-7i
−−−
22
61.3
1,490.2
160.9
2,264.8
169.9
2,423.1
186.1
1,935.7
144.0
2,334.3
140.5


 


EAM170
TGGCATTCACCGCCGTGCCTTA
smallRNA-11
+−−
22
81.2
14,248.4
1,981.3
9,624.9
713.6
10,313.7
1,182.8
10,918.3
1,667.3
10,835.0
1,271.0


 


EAM171
CTACGCGTATTCTTAAGCAATAA
mmu-miR-137
+++
23
64.5
1,447.0
203.0
2,838.4
353.2
2,888.5
362.3
2,198.6
173.6
980.1
149.5


 


EAM172
CTCGTACTGAGCAGGATTA
smallRNA-5
+−−
19
56.9
44.0
4.5
46.8
8.0
12.3
1.7
3.2
0.7
14.5
1.7


 


EAM173
GTCTCGAAAAGGTAGCGTTC
smallRNA-6
+−−
20
63.4
0.4
0.1
0.2
0.1
0.1
0.1
0.1
0.1
0.6
0.2


 


EAM174
AGAAAACATGCTCCAGGTGA
smallRNA-1
+−−
20
64.2
871.5
197.4
1,340.2
391.0
916.4
181.3
583.7
203.8
47.4
15.6


 


EAM175
TCGCCCTCTCAACCCAGCTTTT
mmu-miR-320
+++
22
76.2
1,486.0
215.2
699.9
134.9
600.1
174.5
456.5
83.1
184.9
22.5


 


EAM176
TACAGTACTGTGATAGCTGAA
smallRNA-10
+−−
21
54.5
20.2
2.0
18.8
1.4
26.3
2.0
12.3
1.3
9.6
0.8


 


EAM177
TTCAGCTATCACAGTACTGTA
mmu-miR-101b
+++
21
54.5
192.3
46.0
200.3
30.2
535.9
155.0
373.1
85.0
134.7
18.8


 


EAM178
TGCCAATATTTCTGTGCTGCTA
mmu-miR-195
−−−
22
68.1
1,033.9
229.1
1,350.1
227.0
896.1
165.8
788.6
107.8
407.7
79.7


 


EAM179
ACTATGCAACCTACTACCTCT
mmu-let-7d
+++
21
57.0
4,055.1
310.6
4,266.3
260.2
4,631.8
420.4
4,720.1
316.9
4,959.5
189.5


 


EAM180
AACTATACAATCTACTACCTCA
mmu-let-7f
−−−
22
52.7
3,352.8
273.7
4,133.0
135.2
4,959.5
440.1
4,070.1
267.0
5,438.6
145.6


 


EAM181
AACTATACAATCTACTACCTCA
mmu-let-7f
+++
22
52.7
3,368.5
284.6
3,732.8
323.2
3,508.9
326.6
2,856.4
235.0
3,964.3
116.9


 


EAM182
AACTGTACACACTACTACCTCA
let-7h#
+−−
22
55.5
501.5
33.4
1,100.7
111.9
1,427.3
90.8
1,236.7
138.6
1,493.0
123.4


 


EAM183
AGCACAAACTACTACCTCA
mmu-let-7i
+++
19
53.4
712.3
154.3
1,057.2
145.6
1,355.0
175.8
1,278.7
175.4
1,255.4
182.0


 


EAM184
CACAAGTTCGGATCTACGGGTT
mmu-miR-100
+++
22
69.9
2,064.9
179.2
3,752.3
358.9
2,369.2
159.6
1,721.4
151.9
529.1
70.9


 


EAM185
TCATAGCCCTGTACAATGCTGCT
mmu-miR-103
+++
23
70.8
678.7
71.9
726.4
126.1
930.6
189.4
700.0
87.1
348.2
37.1


 


EAM186
GCTACCTGCACTGTAAGCACTTTT
hsa-miR-106a
++−
24
69.5
2,295.8
374.7
1,801.8
258.6
850.3
131.6
309.4
29.4
72.0
13.3


 


EAM187
TGATAGCCCTGTACAATGCTGCT
mmu-miR-107
+++
23
70.8
653.4
87.6
626.4
109.5
1,589.6
429.9
1,156.6
210.2
316.4
74.2


 


EAM188
AATGCCCCTAAAAATCCTTAT
mir-108&
+−−
21
64.2
3.2
0.7
3.2
0.8
3.7
1.0
2.7
0.6
4.3
1.3


 


EAM189
CACAAATTCGGATCTACAGGGTA
mmu-miR-10a
+++
23
68.0
169.3
28.2
244.4
25.4
251.9
72.6
117.0
13.8
33.7
3.9


 


EAM190
ACAAATTCGGTTCTACAGGGTA
hsa-miR-10b
++−
22
65.3
18.9
3.9
372.6
121.9
115.2
23.6
55.6
15.1
6.9
1.9


 


EAM191
ACAAACACCATTGTCACACTCCA
mmu-miR-122a
+++
23
69.1
20.6
3.4
26.7
3.7
52.5
9.9
34.3
5.8
19.3
3.4


 


EAM192
CGCGTACCAAAAGTAATAATG
mmu-miR-126*
+++
21
63.0
438.1
76.9
1,050.8
139.3
1,443.1
186.3
1,223.7
141.3
511.5
72.2


 


EAM193
CACAGGTTAAAGGGTCTCAGGGA
mmu-miR-125a
+++
23
71.4
3,668.1
159.5
3,116.3
211.8
2,407.7
139.4
2,312.6
157.2
2,991.0
251.2


 


EAM194
AAAAGAGACCGGTTCACTGTGA
mmu-miR-128a
+++
22
67.9
1,766.9
245.8
6,427.0
382.0
7,554.6
951.0
7,463.6
882.0
8,526.6
963.2


 


EAM195
GAAAGAGACCGGTTCACTGTGA
mmu-miR-128b
+++
22
68.2
1,949.8
146.4
4,456.1
503.4
5,246.5
596.9
4,917.9
581.0
6,067.3
385.7


 


EAM196
GCAAGCCCAGACCGAAAAAAG
mmu-miR-129
−−−
21
72.9
0.3
0.1
0.1
0.1
0.2
0.1
0.0
0.1
0.1
0.1


 


EAM197
GCAAGCCCAGACCGCAAAAAG
mmu-miR-129
−−−
21
75.8
22.1
5.1
29.0
4.7
103.4
18.5
114.6
18.8
167.0
30.3


 


EAM198
GCCCTTTCATCATTGCACTG
mmu-miR-130b
+++
20
67.7
679.2
69.0
456.1
41.8
135.4
20.5
32.7
3.8
27.1
6.6


 


EAM199
ACGACCATGGCTGTAGACTGTT
mmu-miR-132
−−−
23
68.2
17.9
4.2
44.5
8.6
60.7
8.7
265.0
45.7
533.7
83.5


 


EAM200
ACAGCTGGTTGAAGGGGACCAA
mmu-miR-133
+++
22
74.5
4.7
1.1
11.0
2.3
10.2
2.4
23.9
4.6
55.0
12.1


 


EAM201
TAGCTGGTTGAAGGGGACCAA
miR-133b%
+−−
21
71.0
4.9
1.0
5.4
0.9
11.0
1.7
27.0
4.7
41.4
9.1


 


EAM202
TCCCTCTGGTCAACCAGTCACA
mmu-miR-134
+++
22
71.4
259.7
46.3
62.1
7.2
136.8
22.8
36.0
5.9
23.0
4.3


 


EAM203
TTCACATAGGAATAAAAAGCCATA
mmu-miR-135
+++
24
65.7
816.6
104.5
885.6
183.3
530.7
73.4
263.8
74.7
159.5
30.9


 


EAM204
ATCACATAGGAATAAAAAGCCATA
mmu-miR-135
−−−
24
64.9
1,181.3
167.5
1,086.4
208.4
759.4
120.7
239.1
59.2
141.1
28.8


 


EAM205
GATTCACAACACCAGCT
mmu-miR-138
+++
17
52.9
486.9
79.7
891.1
137.0
1,702.4
182.5
3,191.6
317.1
1,836.9
167.5


 


EAM206
AGACACGTGCACTGTAGA
mmu-miR-139
+++
18
54.0
55.0
12.3
19.1
3.0
56.0
9.3
189.4
45.4
313.1
58.1


 


EAM207
CTACCATAGGGTAAAACCACT
mmu-miR-140
+++
21
60.1
112.9
22.6
79.0
12.5
142.3
29.1
124.5
22.1
57.5
8.7


 


EAM208
CCATCTTTACCAGACAGTGTT
mmu-miR-141
+++
21
60.7
0.0
0.1
−0.1
0.0
0.0
0.0
0.1
0.1
0.1
0.0


 


EAM209
GTAGTGCTTTCTACTTTATG
mmu-miR-142-5p
+++
20
51.1
40.8
8.6
84.4
15.8
10.0
2.6
48.1
11.2
17.2
4.3


 


EAM210
tgAGCTACAGTGCTTCATCTCA
mmu-miR-143
+++
22
65.0
110.7
27.5
125.8
26.5
252.8
40.1
255.4
41.3
210.1
35.6


 


EAM211
CTAGTACATCATCTATACTGTA
mmu-miR-144
+++
22
48.2
42.4
12.5
230.5
44.6
55.6
8.0
27.2
4.6
34.8
6.6


 


EAM212
AAGGGATTCCTGGGAAAACTGGAC
mmu-miR-145
+++
24
75.1
164.4
34.3
196.0
32.2
121.7
25.1
186.7
32.3
324.8
62.4


 


EAM213
AAACCCATGGAATTCAGTTCTCA
mmu-miR-146
−−−
23
69.5
330.9
35.3
516.9
56.7
1,015.5
145.0
1,291.6
154.9
498.7
93.5


 


EAM214
ACAAAGTTCTGTAGTGCACTGA
mmu-miR-148a
+++
22
61.6
124.1
21.4
87.7
15.1
97.7
16.9
64.8
13.8
49.3
10.3


 


EAM215
ACAAAGTTCTGTGATGCACTGA
mmu-miR-148b
+++
22
64.5
144.9
24.7
142.6
18.7
115.2
15.4
122.7
28.8
82.9
14.9


 


EAM216
GGAGTGAAGACACGGAGCCAGA
mmu-miR-149
+++
22
72.9
834.3
128.0
389.9
43.1
401.0
69.9
301.0
72.3
139.0
25.1


 


EAM217
ACACTGGTACAAGGGTTGGGAGA
mmu-miR-150
+++
23
71.5
11.5
2.9
18.5
2.4
37.0
7.0
210.1
52.0
119.1
25.5


 


EAM218
CCAAGTTCTGTCATGCACTGA
mmu-miR-152
+++
21
65.5
61.3
10.6
35.3
3.4
57.2
12.5
25.1
4.5
17.1
3.1


 


EAM219
TCACTTTTGTGACTATGCAA
mmu-miR-153
+++
20
58.3
374.3
73.4
279.5
65.2
247.4
40.0
147.9
41.1
140.0
36.7


 


EAM220
CGAAGGCAACACGGATAACCTA
mmu-miR-154
+++
22
71.1
84.3
45.0
205.9
63.5
385.0
63.2
400.0
90.7
328.0
79.0


 


EAM221
CCCCTATCACAATTAGCATTAA
mmu-miR-155
+++
22
63.9
16.6
2.5
4.2
0.6
8.7
1.7
5.9
0.9
1.4
0.3


 


EAM222
CACAAACCATTATGTGCTGCTA
mmu-miR-15a
+++
22
65.8
942.0
126.5
932.1
159.8
484.6
84.8
276.3
41.2
202.9
29.0


 


EAM223
TGTAAACCATGATGTGCTGCTA
mmu-miR-15b
+++
22
66.0
1,170.9
162.3
1,266.4
128.1
414.2
52.1
246.1
25.0
250.8
34.9


 


EAM224
ACTACCTGCACTGTAAGCACTTTG
mmu-miR-17-5p
+++
24
67.8
2,138.3
185.2
1,787.0
197.1
742.2
78.9
226.5
22.9
131.2
21.5


 


EAM225
TATCTGCACTAGATGCACCTTA
mmu-miR-18
+++
22
62.4
568.8
90.5
281.4
55.5
222.0
30.4
27.0
3.4
3.6
0.7


 


EAM226
ACTCACCGACAGCGTTGAATGTT
mmu-miR-181a
+++
23
72.6
6,316.3
471.0
3,108.6
187.9
2,126.0
193.9
1,769.8
204.9
1,459.2
175.2


 


EAM227
AACCCACCGACAGCAATGAATGTT
mmu-miR-181b
+++
24
75.8
6,484.5
383.1
2,798.8
281.0
2,081.5
221.8
1,613.1
196.0
1,137.5
128.1


 


EAM228
ACTCACCGACAGGTTGAATGTT
mmu-miR-181c
+++
22
67.6
3,725.6
443.9
2,234.8
149.4
1,144.0
181.6
1,084.7
148.7
924.0
124.3


 


EAM229
TGTGAGTTCTACCATTGCCAAA
mmu-miR-182
+++
22
67.4
34.2
8.4
11.8
2.1
18.5
3.6
53.5
14.5
61.8
14.8


 


EAM230
CAGTGAATTCTACCAGTGCCATA
mmu-miR-183
+++
23
66.5
71.3
20.2
30.7
3.5
81.7
23.1
83.1
14.9
91.3
19.7


 


EAM231
CGGCTGCAACACAAGACACGA
mmu-miR-187
+++
21
74.1
37.2
7.0
29.9
3.5
57.7
8.8
136.4
27.6
48.0
8.4


 


EAM232
GGCTGTCAATTCATAGGTCAG
mmu-miR-192
+++
21
63.7
8.1
2.3
16.7
4.0
10.1
2.5
8.1
1.8
16.8
3.4


 


EAM233
CCCAACAACATGAAACTACCTA
mmu-miR-196
+++
22
64.0
0.9
0.1
0.3
0.1
0.3
0.0
0.3
0.1
0.2
0.1


 


EAM234
GAACAGGTAGTCTGAACACTGGG
mmu-miR-199a
+++
23
67.1
181.6
37.6
123.4
23.9
84.6
18.5
35.3
7.6
12.3
3.0


 


EAM235
GAACAGATAGTCTAAACACTGGG
hsa-miR-199b
++−
23
62.2
98.3
24.1
41.8
6.8
62.3
7.7
27.1
3.4
3.8
0.8


 


EAM236
TCAGTTTTGCATAGATTTGCACA
mmu-miR-19a
+++
23
68.0
1,062.4
192.3
953.5
170.6
442.8
73.0
110.1
16.8
41.5
10.2


 


EAM237
TCAGTTTTGCATGGATTTGCACA
mmu-miR-19b
+++
23
72.9
1,345.4
273.7
1,068.5
230.3
631.6
91.3
147.6
24.3
34.7
6.3


 


EAM238
ATACATACTTCTTTACATTCCA
mmu-miR-1
+++
22
56.0
36.0
13.0
95.8
18.4
171.8
54.0
159.4
22.7
50.2
10.6


 


EAM239
ATACATACTTCTTTACATTCCA
mmu-miR-1
−−−
22
56.0
21.5
6.0
77.8
20.6
136.2
40.8
189.5
34.6
44.8
7.4


 


EAM240
CTACCTGCACTATAAGCACTTTA
mmu-miR-20
+++
23
61.7
2,300.0
254.6
831.7
74.1
446.0
71.4
107.3
21.7
54.3
11.3


 


EAM241
CTAGTGGTCCTAAACATTTCAC
mmu-miR-203
+++
22
60.5
20.2
4.5
17.5
2.4
21.8
4.9
10.7
2.2
3.9
0.5


 


EAM242
AGGCATAGGATGACAAAGGGAA
mmu-miR-204
+++
22
69.6
160.6
38.5
187.1
33.0
310.1
56.6
349.2
64.2
188.7
40.1


 


EAM243
CAGACTCCGGTGGAATGAAGGA
mmu-miR-205
+++
22
72.7
16.1
3.5
4.4
0.7
3.9
0.9
2.7
0.3
1.5
0.2


 


EAM244
TCAACATCAGTCTGATAAGCTA
mmu-miR-21
+++
22
59.4
317.1
73.9
211.1
48.5
250.8
32.0
183.6
32.2
140.4
34.6


 


EAM245
CAGCCGCTGTCACACGCACAG
mmu-miR-210
+++
21
77.0
331.4
53.8
193.3
34.6
97.2
13.0
49.9
5.7
44.9
8.9


 


EAM246
AGGCGAAGGATGACAAAGGGAA
hsa-miR-211
++−
22
73.9
78.1
21.8
142.6
26.3
140.1
21.9
189.3
45.0
105.7
24.4


 


EAM247
GGCCGTGACTGGAGACTGTTA
mmu-miR-212
+++
21
68.9
4.9
0.9
6.4
1.5
7.9
1.3
49.0
11.5
70.3
17.1


 


EAM248
GGTACAATCAACGGTCGATGGT
mmu-miR-213
+++
22
70.3
280.5
63.2
181.0
38.4
244.3
67.7
100.0
18.6
33.1
5.2


 


EAM249
CTGCCTGTCTGTGCCTGCTGT
mmu-miR-214
+++
21
72.4
231.8
39.5
27.1
2.6
7.5
0.6
5.7
0.8
2.4
0.3


 


EAM250
GTCTGTCAATTCATAGGTCAT
hsa-miR-215
++−
21
57.6
12.8
2.7
9.3
1.1
14.5
3.5
14.5
2.7
9.0
1.4


 


EAM251
CACAGTTGCCAGCTGAGATTA
mmu-miR-216
+++
21
65.4
37.9
7.0
16.6
2.3
13.2
1.7
12.2
2.6
3.1
0.3


 


EAM252
ATCCAATCAGTTCCTGATGCAGTA
hsa-miR-217
++−
24
69.7
14.5
3.3
5.1
0.6
12.9
2.2
10.4
2.2
0.3
0.1


 


EAM253
ACATGGTTAGATCAAGCACAA
mmu-miR-218
+++
21
62.1
156.6
35.7
201.8
46.4
174.8
31.6
251.0
61.0
256.5
56.7


 


EAM254
AGAATTGCGTTTGGACAATCA
mmu-miR-219
+++
21
67.3
808.0
131.8
374.5
65.2
259.9
44.2
1,626.7
187.8
378.0
76.4


 


EAM255
ACAGTTCTTCAACTGGCAGCTT
mmu-miR-22
+++
22
67.6
187.2
48.4
187.7
35.6
489.7
61.1
528.5
112.3
567.0
117.9


 


EAM256
AAAGTGTCAGATACGGTGTGG
hsa-miR-220
++−
21
63.9
0.9
0.2
0.8
0.2
0.4
0.2
0.4
0.0
0.5
0.1


 


EAM257
GAAACCCAGCAGACAATGTAGCT
mmu-miR-221
+++
23
69.5
167.9
42.7
165.1
29.9
548.1
118.1
436.5
57.5
321.8
65.6


 


EAM258
GAGACCCAGTAGCCAGATGTAGCT
mmu-miR-222
+++
24
70.3
12.2
1.6
14.3
1.2
63.0
5.8
128.0
19.5
43.3
8.4


 


EAM259
GGGGTATTTGACAAACTGACA
mmu-miR-223
+++
21
64.0
22.7
4.8
26.4
5.0
27.0
3.9
18.7
3.8
7.2
1.9


 


EAM260
GGAAATCCCTGGCAATGTGAT
mmu-miR-23a
+++
21
70.2
728.6
185.5
407.2
76.8
451.0
101.0
742.4
117.1
799.5
118.5


 


EAM261
GTGGTAATCCCTGGCAATGTGAT
mmu-miR-23b
+++
23
71.8
383.9
67.5
377.2
64.9
286.6
49.7
730.2
178.5
780.1
64.6


 


EAM262
CTGTTCCTGCTGAACTGAGCCA
mmu-miR-24
+++
22
71.8
884.8
169.5
505.3
74.4
538.2
94.0
1,248.9
236.4
1,379.9
246.6


 


EAM263
AGCCTATCCTGGATTACTTGAA
mmu-miR-26a
+++
22
64.7
1,684.2
121.7
1,926.5
151.5
1,894.7
83.0
1,903.1
204.2
2,120.1
206.0


 


EAM264
CAGAACTTAGCCACTGTGAA
mmu-miR-27b
+++
20
59.8
190.9
36.1
144.2
26.2
237.1
38.3
246.7
58.3
472.7
96.6


 


EAM265
AACCGATTTCAGATGGTGCTAG
mmu-miR-29a
−−−
22
67.9
151.0
29.1
287.7
40.1
554.9
66.4
2,232.4
213.9
3,477.8
188.2


 


EAM266
AACACTGATTTCAAATGGTGCTA
mmu-miR-29b
−−−
23
66.2
103.7
18.3
95.4
11.4
333.1
70.3
3,050.7
267.3
4,065.9
504.7


 


EAM267
AACACTGATTTCAAATGGTGCTA
mmu-miR-29b
−−−
23
66.2
72.6
14.9
98.1
15.0
385.3
71.5
2,762.9
345.3
3,533.7
372.0


 


EAM268
AACCGATTTCAGATGGTGCTAG
mmu-miR-29a
+++
22
67.9
88.4
15.9
129.6
13.2
318.4
62.8
1,923.0
222.4
2,658.1
385.3


 


EAM269
GCTGCAAACATCCGACTGAAAG
mmu-miR-30a
−−−
22
71.4
223.0
43.0
242.0
37.9
214.0
25.5
303.8
56.5
146.7
23.5


 


EAM270
GCTGAGTGTAGGATGTTTACA
mmu-miR-30b
+++
21
59.2
889.3
112.0
877.1
122.9
626.9
67.2
840.1
108.9
781.5
156.8


 


EAM271
GCTGAGAGTGTAGGATGTTTACA
mmu-miR-30c
+++
23
63.3
899.5
177.8
1,225.9
201.3
988.4
125.5
887.5
103.1
881.8
134.2


 


EAM272
CTTCCAGTCGGGGATGTTTACA
mmu-miR-30d
+++
22
70.6
1,704.5
259.1
1,140.1
144.7
959.6
171.7
1,062.6
150.7
1,274.8
174.9


 


EAM273
CAATGCAACTACAATGCAC
mmu-miR-33
+++
19
58.7
1,055.8
126.7
2,245.2
338.3
2,627.0
96.2
1,951.9
255.9
518.7
51.9


 


EAM274
CAATGCAACAGCAATGCAC
miR-33b%
+−−
19
65.0
59.6
9.7
125.4
22.9
193.2
27.2
61.7
9.7
58.9
8.2


 


EAM275
ACAACCAGCTAAGACACTGCCA
mmu-miR-34a
+++
22
69.0
319.3
40.8
289.8
45.6
320.7
24.2
258.5
46.1
404.1
82.3


 


EAM276
TCATACAGCTAGATAACCAAAGA
mmu-miR-9
+++
23
61.4
6,168.0
683.6
4,628.0
272.5
3,281.3
413.8
2,797.7
447.1
2,336.5
462.2


 


EAM277
GCAAAAATGTGCTAGTGCCAAA
mmu-miR-96
+++
22
70.0
12.1
2.9
10.3
2.5
14.0
2.8
23.1
6.0
41.1
10.0


 


EAM278
AACAATACAACTTACTACCTCA
mmu-miR-98
+++
22
55.9
318.4
30.7
514.7
90.4
653.3
103.1
526.8
74.3
410.8
71.0


 


EAM279
TAACCGATTTCAAATGGTGCTA
mmu-miR-29c
+++
22
67.2
131.8
30.4
198.4
41.0
504.3
49.7
2,582.6
317.8
3,964.0
178.6


 


EAM280
GCTGCAAACATCCGACTGAAAG
mmu-miR-30a
+++
22
71.4
262.3
52.0
209.7
30.8
188.2
37.9
391.7
65.2
139.1
26.6


 


EAM281
ATCCAGTCAGTTCCTGATGCAGTA
mmu-miR-217
+++
24
70.3
14.7
3.1
8.1
1.3
18.1
2.8
7.7
1.3
0.8
0.2


 


EAM282
GAACAGGTAGTCTAAACACTGGG
mmu-miR-199b
+++
23
64.5
193.5
47.1
87.9
18.1
62.8
9.2
30.9
6.0
3.7
0.9


 


EAM283
AGGCAAAGGATGACAAAGGGAA
mmu-miR-211
+++
22
72.5
138.4
29.7
325.1
61.1
466.0
60.7
303.6
57.7
192.8
33.8


 


EAM284
AGAAAACATGCTCCAGGTGA
smallRNA-1
−−−
20
64.2
910.9
229.1
793.5
160.3
905.0
269.5
476.7
75.5
40.9
6.8


 


EAM285
ACTGGAGACACGTGCACTGTAGA
miR-139#
−−−
23
68.8
68.4
13.5
56.3
10.9
54.3
7.9
374.6
91.2
376.3
46.2


 


EAM286
TTAAATTAACCGCGAATTCGC
smallRNA-7
+−−
21
69.4
4.5
1.2
3.0
0.5
3.5
0.7
1.3
0.2
2.4
0.5


 


EAM287
AAGACGGTGCTTACCTGTTCC
smallRNA-8
+−−
21
67.6
13.5
2.8
9.7
1.5
22.4
2.9
8.8
1.1
6.4
1.0


 


EAM288
ACACAAATTCGGTTCTACAGGG
mmu-miR-10b
+++
22
67.7
9.5
1.4
184.8
31.5
106.7
17.1
48.6
6.5
6.3
0.9


 


EAM289
AACAAGCCCAGACCGCAAAAAG
mmu-miR-129
+++
22
74.6
18.5
3.5
23.7
2.3
70.5
15.8
73.6
13.4
95.8
15.7


 


EAM290
ACCCTTATCAGTTCTCCGTCCA
mmu-miR-184
+++
22
69.4
2.7
0.5
1.6
0.1
1.9
0.6
3.0
0.9
2.7
0.8


 


EAM291
GAACTGCCTTTCTCTCCA
mmu-miR-185
+++
18
58.6
664.4
130.9
493.0
111.3
428.3
67.3
963.1
123.9
447.2
69.7


 


EAM292
AAGCCCAAAAGGAGAATTCTTTG
mmu-miR-186
+++
23
70.5
178.6
35.5
101.7
16.5
102.6
11.6
101.3
14.7
59.9
12.0


 


EAM293
ACCCTCCACCATGCAAGGGATG
mmu-miR-188
+++
22
76.7
26.5
3.9
35.6
7.7
94.3
25.3
21.4
5.0
8.5
1.4


 


EAM294
ACTGATGTCAGCTCAGTAGGCAC
mmu-miR-189
+++
23
67.7
12.0
2.2
9.8
1.8
20.2
5.1
20.1
3.5
14.5
3.4


 


EAM295
ACCTAATATATCAAACATATCA
mmu-miR-190
+++
22
53.7
41.5
9.1
37.6
6.0
63.7
13.5
41.3
5.4
39.7
7.1


 


EAM296
AGCTGCTTTTGGGATTCCGTTG
mmu-miR-191
+++
22
74.5
2,769.9
200.2
2,792.5
302.4
2,650.5
114.3
2,470.8
297.4
2,153.6
184.4


 


EAM297
CTGGGACTTTGTAGGCCAGTT
mmu-miR-193
+++
21
67.6
397.2
54.3
562.8
49.3
388.2
44.6
512.0
64.7
294.4
32.9


 


EAM298
TCCACATGGAGTTGCTGTTACA
mmu-miR-194
+++
22
67.9
16.4
4.6
20.2
4.4
15.4
2.6
28.1
6.2
11.2
3.4


 


EAM299
GCCAATATTTCTGTGCTGCTA
mmu-miR-195
+++
21
65.2
768.1
124.8
820.3
125.9
479.0
48.8
472.3
75.1
534.4
73.2


 


EAM300
GCTGGGTGGAGAAGGTGGTGAA
hsa-miR-197
++−
22
74.9
3.2
0.5
2.1
0.3
1.0
0.2
1.2
0.2
1.8
0.3


 


EAM301
CCTATCTCCCCTCTGGACC
hsa-miR-198
++−
19
64.5
0.8
0.3
0.2
0.1
3.0
0.9
0.3
0.1
1.1
0.2


 


EAM302
AACAGGTAGTCTGAACACTGGG
mmu-miR-199a
−−−
22
65.0
228.3
35.3
111.4
15.8
62.9
10.0
23.0
4.7
12.1
2.3


 


EAM303
AACCAATGTGCAGACTACTGTA
mmu-miR-199a*
+++
22
61.7
203.0
29.9
111.5
12.4
76.3
12.2
19.6
2.1
11.0
1.5


 


EAM304
CATCGTTACCAGACAGTGTTA
mmu-miR-200a
+++
21
59.9
19.0
7.0
60.4
11.4
295.4
39.7
515.8
83.6
242.9
66.7


 


EAM305
GTCATCATTACCAGGCAGTATTA
mmu-miR-200b
+++
23
63.5
19.2
4.0
14.8
1.7
97.3
20.5
110.9
22.8
87.8
13.7


 


EAM306
AGAACAATGCCTTACTGAGTA
mmu-miR-201
+++
21
58.6
0.1
0.2
0.5
0.4
0.0
0.1
−0.2
0.2
−0.3
0.2


 


EAM307
TCTTCCCATGCGCTATACCTCT
mmu-miR-202
+++
22
69.9
0.2
0.1
0.3
0.1
0.2
0.1
0.4
0.1
0.2
0.0


 


EAM308
CCACACACTTCCTTACATTCCA
mmu-miR-206
+++
22
66.6
59.4
15.1
118.9
27.5
300.4
48.1
672.6
118.0
310.3
40.3


 


EAM309
GAGGGAGGAGAGCCAGGAGAAGC
mmu-miR-207
+++
23
76.0
3.4
1.0
1.2
0.3
0.9
0.2
0.7
0.1
1.6
0.3


 


EAM310
ACAAGCTTTTTGCTCGTCTTAT
mmu-miR-208
+++
22
65.3
−0.1
0.2
1.3
0.5
1.0
0.5
0.7
0.2
0.4
0.2




















TABLE 3









SEQ ID



Oligo ID
Oligo sequence
NO:




















EAM101
TCCATCATCAAAACAAATGGAGT
5



EAM102
TCCATCATGAAAAGAAATGGAGT
6



EAM103
TGGCATTCACCGCGTGCCTTA
7



EAM104
TGGCATTCAGCGGGTGCCTTA
8



EAM105
TCACAAGTTAGGGTCTCAGGGA
9



EAM106
TCACAAGTAAGGGTGTCAGGGA
10



EAM107
TGTTCCTGCTGAACTGAGCCA
11



EAM108
TGTTCCTGGTGAAGTGAGCCA
12



EAM109
AACAACAAAATCACTAGTCTTCCA
13



EAM110
AACAACAAAATGAGTAGTCTTCCA
14



EAM1100
GCATGCATGCATGCATGCATG
15



EAM1101
GTGGTAGCGCAGTGCGTAGAA
16



EAM1102
GGTGATGCCCTGAATGTTGTC
17



EAM1103
TGTCATGGATGACCTTGGCCA
18



EAM1104
CTTTTGACATTGAAGGGAGCT
19



EAM111
TAACTGTACAAACTACTACCTCA
20



EAM112
TAACTGTAGAAAGTACTACCTCA
21



EAM113
ACAGGTTAAAGGGTCTCAGGGA
22



EAM114
ACAGGTAAAAGGGTGTCAGGGA
23



EAM115
CGCCAATATTTACGTGCTGCTA
24



EAM116
CGCCAATATTAAGGTGCTGCTA
25



EAM117
TAACCGATTTCAAATGGTGCTA
26



EAM118
TAACCGATTTGAAAAGGTGCTA
27



EAM119
AACACTGATTTCAAATGGTGCTA
28



EAM120
AACACTGATTTGAAAAGGTGCTA
29



EAM121
CACAAGATCGGATCTACGGGT
30



EAM122
CACAAGATGGGATGTACGGGT
31



EAM123
AACTATGCAACCTACTACCTCT
32



EAM124
AACTATGCAACGTAGTACCTCT
33



EAM125
CGGCCTGATTCACAACACCAGCT
34



EAM126
CGGCCTGATTGAGAACACCAGCT
35



EAM127
TCATAGCCCTGTACAATGCTGCT
36



EAM128
TCATAGCCCTGAAGAATGCTGCT
37



EAM129
AGGCATTCACCGCGTGCCTTAT
38



EAM130
AGGCATTCAGCGGGTGCCTTAT
39



EAM131
ACAGGCCGGGACAAGTGCAATAT
40



EAM132
ACAGGCCGGGAGAAGAGCAATAT
41



EAM133
ACACCAATGCCCTAGGGGATGCG
42



EAM134
ACACCAATGGCGTAGGGGATGCG
43



EAM135
CAACAAACATTTAATGAGGCC
44



EAM136
CAACAAAGATTAAATGAGGCC
45



EAM137
CCGACCATGGCTGTAGACTGTTA
46



EAM138
CCGACCATGGGTGAAGACTGTTA
47



EAM139
TAACCCATGGAATTCAGTTCTCA
48



EAM140
TAACCCATGGAAATGAGTTCTCA
49



EAM141
TAACTATACAATCTACTACCTCA
50



EAM142
TAACTATACAATGTAGTACCTCA
51



EAM143
TAACCATACAACCTATTACCTCA
52



EAM144
TAACCATAGAACGTATTACCTCA
53



EAM145
AACCATACAACCTACTACCTCA
54



EAM146
AACCATACAAGCTAGTACCTCA
55



EAM147
AACCACACAACCTACTACCTCA
56



EAM148
AACCACACAAGCTAGTACCTCA
57



EAM149II
GCATTCACCCGCGTGCCTTA
58



EAM150II
TCATACAGCTAGATAACCAAAGA
59



EAM151
ACAAGATCGGATCTACGG
60



EAM152
ACTTTCGGTTATCTAGCTTTAT
61



EAM153
AACTATACAACCTACTACCTCA
62



EAM154
AAAGAGACCGGTTCACTGTGA
63



EAM155
TCCATCATCAAAACAAATGGAGT
64



EAM156
ACTCACCGAGAGAGCGTTGAATGTT
65



EAM157
TGGTGTCAGAAGTGGGATAC
66



EAM158
TACAGCTAAATAACCAAAGA
67



EAM159
ATGCCCTTTTAACATTGCACTG
68



EAM160
AACCTATCCTGAATTACTTGAA
69



EAM161
CTCAATAGACTGTGAGCTCCTT
70



EAM162
ATCAAGGTCCGCTGTGAACACG
71



EAM163
TCCATAAAGTAGGAAACACTACA
72



EAM164
GAGTGCTTGCTAGGTGCCAAG
73



EAM165
GGGAGTGAAGACACGGAGCCAGA
74



EAM166
GCCTATCCTGGATTACTTGAA
75



EAM167
GTTGTGGTCACTTACAATT
76



EAM168
CTATACAACCTCCTACCTCA
77



EAM169
AACAGCACAAACTACTACCTCA
78



EAM170
TGGCATTCACCGCCGTGCCTTA
79



EAM171
CTACGCGTATTCTTAAGCAATAA
80



EAM172
CTCGTACTGAGCAGGATTA
81



EAM173
GTCTCGAAAAGGTAGCGTTC
82



EAM174
AGAAAACATGCTCCAGGTGA
83



EAM175
TCGCCCTCTCAACCCAGCTTTT
84



EAM176
TACAGTACTGTGATAGCTGAA
85



EAM177
TTCAGCTATCACAGTACTGTA
86



EAM178
TGCCAATATTTCTGTGCTGCTA
87



EAM179
ACTATGCAACCTACTACCTCT
88



EAM180
AACTATAGAATCTACTACCTCA
89



EAM181
AACTATACAATCTACTACCTCA
90



EAM182
AACTGTACACACTACTACCTCA
91



EAM183
AGCACAAACTACTACCTCA
92



EAM184
CACAAGTTCGGATCTACGGGTT
93



EAM185
TCATAGCCCTGTACAATGCTGCT
94



EAM186
GCTACCTGCACTGTAAGCACTTTT
95



EAM187
TGATAGCCCTGTACAATGCTGCT
96



EAM188
AATGCCCCTAAAAATCCTTAT
97



EAM189
CACAAATTCGGATCTACAGGGTA
98



EAM190
ACAAATTCGGTTCTACAGGGTA
99



EAM191
ACAAACACCATTGTCACACTCCA
100



EAM192
CGCGTACCAAAAGTAATAATG
101



EAM193
CACAGGTTAAAGGGTCTCAGGGA
102



EAM194
AAAAGAGACCGGTTCACTGTGA
103



EAM195
GAAAGAGACCGGTTCACTGTGA
104



EAM196
GCAAGCCCAGACCGAAAAAAG
105



EAM197
GCAAGCCCAGACCGCAAAAAG
106



EAM198
GCCCTTTCATCATTGCACTG
107



EAM199
ACGACCATGGCTGTAGACTGTTA
108



EAM200
ACAGCTGGTTGAAGGGGACCAA
109



EAM201
TAGGTGGTTGAAGGGGACCAA
110



EAM202
TCCCTCTGGTCAACCAGTCACA
111



EAM203
TTCACATAGGAATAAAAAGCCATA
112



EAM204
ATCACATAGGAATAAAAAGCCATA
113



EAM205
GATTCACAACACCAGCT
114



EAM206
AGACACGTGCACTGTAGA
115



EAM207
CTACCATAGGGTAAAACCACT
116



EAM208
CCATCTTTACCAGACAGTGTT
117



EAM209
GTAGTGCTTTCTACTTTATG
118



EAM210
tgAGCTACAGTGCTTCATCTCA
119



EAM211
CTAGTACATCATCTATACTGTA
120



EAM212
AAGGGATTCCTGGGAAAACTGGAC
121



EAM213
AAACCCATGGAATTCAGTTCTCA
122



EAM214
ACAAAGTTCTGTAGTGCACTGA
123



EAM215
ACAAAGTTCTGTGATGCACTGA
124



EAM216
GGAGTGAAGACACGGAGCCAGA
125



EAM217
ACACTGGTACAAGGGTTGGGAGA
126



EAM218
CCAAGTTCTGTCATGCACTGA
127



EAM219
TCACTTTTGTGACTATGCAA
128



EAM220
CGAAGGCAACACGGATAACCTA
129



EAM221
CCCCTATCACAATTAGCATTAA
130



EAM222
CACAAACCATTATGTGCTGCTA
131



EAM223
TGTAAACCATGATGTGCTGCTA
132



EAM224
ACTACCTGCACTGTAAGCACTTTG
133



EAM225
TATCTGCACTAGATGCACCTTA
134



EAM226
ACTCACCGACAGCGTTGAATGTT
135



EAM227
AACCCACCGACAGCAATGAATGTT
136



EAM228
ACTCACCGACAGGTTGAATGTT
137



EAM229
TGTGAGTTCTACCATTGCCAAA
138



EAM230
CAGTGAATTCTACCAGTGCCATA
139



EAM231
CGGCTGCAACACAAGACACGA
140



EAM232
GGCTGTCAATTCATAGGTCAG
141



EAM233
CCCAACAACATGAAACTACCTA
142



EAM234
GAACAGGTAGTCTGAACACTGGG
143



EAM235
GAACAGATAGTCTAAACACTGGG
144



EAM236
TCAGTTTTGCATAGATTTGCACA
145



EAM237
TCAGTTTTGCATGCATTTGCACA
146



EAM238
ATACATACTTCTTTACATTCCA
147



EAM239
ATACATACTTCTTTACATTCCA
148



EAM240
CTACCTGCACTATAAGCACTTTA
149



EAM241
CTAGTGGTCCTAAACATTTCAC
150



EAM242
AGGCATAGGATGACAAAGGGAA
151



EAM243
CAGACTCCGGTGGAATGAAGGA
152



EAM244
TCAACATCAGTCTGATAAGCTA
153



EAM245
CAGCCGCTGTCACACGCACAG
154



EAM246
AGGCGAAGGATGACAAAGGGAA
155



EAM247
GGCCGTGACTGGAGACTGTTA
156



EAM248
GGTACAATCAACGGTCGATGGT
157



EAM249
CTGCCTGTCTGTGCCTGCTGT
158



EAM250
GTCTGTCAATTCATAGGTCAT
159



EAM251
CACAGTTGCCAGCTGAGATTA
160



EAM252
ATCCAATCAGTTCCTGATGCAGTA
161



EAM253
ACATGGTTAGATCAAGCACAA
162



EAM254
AGAATTGCGTTTGGACAATCA
163



EAM255
ACAGTTCTTCAACTGGCAGCTT
164



EAM256
AAAGTGTCAGATACGGTGTGG
165



EAM257
GAAACCCAGCAGACAATGTAGCT
166



EAM258
GAGACCCAGTAGCCAGATGTAGCT
167



EAM259
GGGGTATTTGACAAACTGACA
168



EAM260
GGAAATCCCTGGCAATGTGAT
169



EAM261
GTGGTAATCCCTGGCAATGTGAT
170



EAM262
CTGTTCCTGCTGAACTGAGCCA
171



EAM263
AGCCTATCCTGGATTACTTGAA
172



EAM264
CAGAACTTAGCCACTGTGAA
173



EAM265
AACCGATTTCAGATGGTGCTAG
174



EAM266
AACACTGATTTCAAATGGTGCTA
175



EAM267
AACACTGATTTCAAATGGTGCTA
176



EAM268
AACCGATTTCAGATGGTGCTAG
177



EAM269
GCTGCAAACATCCGACTGAAAG
178



EAM270
GCTGAGTGTAGGATGTTTACA
179



EAM271
GCTGAGAGTGTAGGATGTTTACA
180



EAM272
CTTCCAGTCGGGGATGTTTACA
181



EAM273
CAATGCAACTACAATGCAC
182



EAM274
CAATGCAACAGCAATGCAC
183



EAM275
ACAACCAGCTAAGACACTGCCA
184



EAM276
TCATACAGCTAGATAACCAAAGA
185



EAM277
GCAAAAATGTGCTAGTGCCAAA
186



EAM278
AACAATACAACTTACTACCTCA
187



EAM279
TAACCGATTTCAAATGGTGCTA
188



EAM280
GCTGCAAACATCCGACTGAAAG
189



EAM281
ATCCAGTCAGTTCCTGATGCAGTA
190



EAM282
GAACAGGTAGTCTAAACACTGGG
191



EAM283
AGGCAAAGGATGACAAAGGGAA
192



EAM284
AGAAAACATGCTCCAGGTGA
193



EAM285
ACTGGAGACACGTGCACTGTAGA
194



EAM286
TTAAATTAACCGCGAATTCGC
195



EAM287
AAGACGGTGCTTACCTGTTCC
196



EAM288
ACACAAATTCGGTTCTACAGGG
197



EAM289
AACAAGCCCAGACCGCAAAAAG
198



EAM290
ACCCTTATCAGTTCTCCGTCCA
199



EAM291
GAACTGCCTTTCTCTCCA
200



EAM292
AAGCCCAAAAGGAGAATTCTTTG
201



EAM293
ACCCTCCACCATGCAAGGGATG
202



EAM294
ACTGATGTCAGCTCAGTAGGCAC
203



EAM295
ACCTAATATATCAAACATATCA
204



EAM296
AGCTGCTTTTGGGATTCCGTTG
205



EAM297
CTGGGACTTTGTAGGCCAGTT
206



EAM298
TCCACATGGAGTTGCTGTTACA
207



EAM299
GCCAATATTTCTGTGCTGCTA
208



EAM300
GCTGGGTGGAGAAGGTGGTGAA
209



EAM301
CCTATCTCCCCTCTGGACC
210



EAM302
AACAGGTAGTCTGAACACTGGG
211



EAM303
AACCAATGTGCAGACTACTGTA
212



EAM304
CATCGTTACCAGACAGTGTTA
213



EAM305
GTCATCATTACCAGGCAGTATTA
214



EAM306
AGAACAATGCCTTACTGAGTA
215



EAM307
TCTTCCCATGCGCTATACCTCT
216



EAM308
CCACACACTTCCTTACATTCCA
217



EAM309
GAGGGAGGAGAGCCAGGAGAAGC
218



EAM310
ACAAGCTTTTTGCTCGTCTTAT
219










A summary of the microarray data is presented in Table 2. Oligonucleotide sequences correspond to probes on the array. MicroRNA names were obtained from the January 2004 release of the Rfam database or if these were not available, then microarray names were obtained from NCBI. Probes named smallRNA-1 through -13 correspond to unique small RNAs that we cloned. The small RNAs did not correspond to known microRNAs and did not have perfect matches in the current release of the rat genome sequence. Column A indicates whether the probe is complementary to a microRNA or to one of the small RNAs we cloned (“−”=no, “+”=yes). Column B indicates whether the probe is complementary to a mouse microRNA. Oligonucleotides with a “−” in column A were either controls or were sequences that while submitted to public databases as microRNAs were later found not to encode microRNAs. In a few cases we printed probes that represented the same microRNA twice, but we analyzed the data from only one of these probes. The probes we did not analyze have no labels in columns A, B, and C. Melting temperatures were calculated using the nearest neighbors method (Breslauer et al., Proc Natl Acad Sci USA 1986, 83:3746-3750). Data for the five time points of mouse brain development (E12.5, E17.5, P4, P18 and adult) are shown. Microarray data was derived as described in more detail below. Briefly, data corresponding to mean spot intensities were averaged over quadruplicates (on each array) and four independent hybridizations. SEM refers to the standard error of the mean. microRNAs labeled with % are not in the current release of Rfam, but are deposited in NCBI and are described elsewhere (Lim et al., Science 299:1540, 2003; Lagos-Quintana et al., Curr Biol 12:735-739, 2002; Dostie RNA 9:180-186, 2003).
Printing and hybridization were done using the protocols from the slides manufacturer with the following modifications: the oligonucleotide concentration for printing was 20 μM in 150 mM sodium phosphate, pH 8.5, and hybridization was at 50° C. for 6 hours. Printing was done using a MicroGrid TAS II arrayer (BIOROBOTICS, Cambridge, UK) at 50% humidity.
Sample and Probe Preparation
Whole brains from three to eight C57BL/6 mice were pooled. Starting with 250 μg of total RNA for each timepoint, 18-26 nucleotide RNA was purified using denaturing polyacrylamide gel electrophoresis. The samples were divided, and the following cloning steps were done twice independently for each timepoint.
3′ and 5′ adaptor oligonucleotides were ligated to 18-26 nucleotide RNA followed by reverse transcription, essentially as described for microRNA cloning (Lagos-Quintana et al., Curr Biol 12:735-739, 2002). Briefly, an RNA-DNA hybrid 5′-pUUUaaccgcgaattccagt-idT-3′ (SEQ ID NO:220) (DHARMACON, Lafayette, Colo.) (X=RNA, x=DNA, p=phosphate, idT=inverted [3′-3′ bond] deoxythymidine) was ligated to the 3′ end and 5′-acggaattcctcactAAA-3′ (SEQ ID NO:221) (DHARMACON, Lafayette, Colo.) was ligated to the 5′ end. The ligation products were divided into two aliquots, and the following steps were done twice independently for each time point. Ligation products were reverse transcribed and amplified by ten rounds of PCR (40 seconds at 94° C., 30 seconds at 50° C., 30 seconds at 72° C.). For PCR, the oligonucleotides used were: oligol: 5′-Cy3-acggaattcctcactaaa-3′ (SEQ ID NO:386) and oligo2: 5′-tactggaattcgcggttaa-3′ (SEQ ID NO:387). The PCR product was precipitated, washed and resuspended in hybridization buffer (5×SSC, 0.1% SDS, 0.1 mg/ml sheared denatured salmon sperm DNA).
Data Acquisition and Analysis
Microarray slides were scanned using an arrayWoRxe biochip reader (APPLIED PRECISION, Issaquah, Wash.), and primary data were analyzed using the Digital Genome System suite (Molecularware, Cambridge, Mass.) and the Spotfire Decision Site (Spotfire, Somerville, Mass.). Cluster analysis was performed using the CLUSTER/TreeView software (Eisen et al., Proc Natl Acad Sci USA 95:14863-14868, 1998).
Samples/Hybridizations
Samples were processed as depicted in FIG. 10. For each sample, two independent ligations were performed. The products of the two ligations were split, and two independent reverse transcription/amplifications/hybridizations were performed. Thus, for each sample data were collected from four independent array hybridizations.
Arrays/Normalization
Glass slides were arrayed using quadruplicate spots for each of the 138 microRNA probes, 19 small RNA probes, and control probes (FIG. 10 and Table 2). A sample microarray scan is shown in FIG. 11. The microarray consists of 16 squares of eight by seven spots each. Quadruplicates are identified as rows of four spots (every other spot) in the individual squares. A TIFF file of the scanned array is used for subsequent array analysis (DIGITAL GENOME SYSTEM SUITE, MOLECULARWARE, Cambridge, Mass.). Normalized spot intensities were used for all data analysis. For inter-array comparisons, all data were scaled based on total array intensities (scaling factors ranged from 0.4 to 2.6), and data for each sample and each gene were averaged and the standard error of the mean (SEM) was calculated. Total array intensity was calculated as the sum of the normalized spot intensity for all spots in the microarray. A variance analysis (ANOVA) was performed using Spotfire DecisionSite (Spotfire). Hierarchical clustering was performed using CLUSTER 3.0/TreeView software (Hoon et al., Bioinformatics 20: 1453-1454, 2004). For CLUSTER 3.0/TreeView output see FIG. 11, which shows a profile of microRNA expression in the developing mouse brain. The gray scale arrows indicate relative signal intensities. The microRNA expression profile was sorted using a hierarchical clustering method (see above). Only data from 66 probes that changed at least two-fold over the developmental time course (ANOVA, P<0.001) are shown. The data used for the analysis are present in Table 2.
Control Probes
Control probes were either negative controls or mismatch controls. The negative controls were either a synthetic (GCAT)n oligonucleotide (EAM100) or sequences derived from mouse mRNA sequences (EAM1101-1104).












oligo ID
oligo sequence
mRNA







EAM1100
GCATGCATGCATGCATGCATG
Synthetic




(SEQ ID NO:222)


 


EAM1101
GTGGTAGCGCAGTGCGTAGAA
beta-tubulin




(SEQ ID NO:223)


 


EAM1102
GGTGATGCCCTGAATGTTGTC
histone H4




(SEQ ID NO:224)


 


EAM1103
TGTCATGGATGACCTTGGCCA
glyceraldehyde




dehydrogenase




(SEQ ID NO:225)


 


EAM1104
CTTTTGACATTGAAGGGAGCT
laminin alpha 4




(SEQ ID NO:226)









The mismatch controls were probes with two mismatches to a specific microRNA probe. For each mismatch control probe, two central Cs were replaced by two Gs; if this change was not possible, one C was replaced with a G and one T with an A. Averaged, normalized spot intensities for these negative controls ranged from −0.8 to 1.2, as compared with 90 to 14,250 for microRNA probes that were scored as signals. Negative values resulted from the subtraction of the local background signal surrounding each spot on the arrays.

Expression Levels and Microarray Correlations

Two methods were used to distinguish signal versus noise. First, we used correlation analysis among the four hybridizations for a given time point to assess reproducibility. Second, we used a set of negative control probes (see above) to measure noise. As an example, FIG. 13 shows the correlations (scatter plots) among the four hybridizations for time point E12.5. For each graph the axes show averaged mean spot intensities for all probes from a given data set, as indicated. Arbitrarily, we chose a cutoff of 90 for our analysis. Expressed relative to background values, a microRNA was identified as being present only if the signal was at least 75-fold over that of the negative controls for at least one timepoint. FIG. 14 shows the correlations (scatter plots) of the data for E12.5 with the data from each other timepoint (each averaged over four hybridizations). As expected, the correlation between E12.5 and E17.5 is highest, and the correlation decreases with samples from more distant developmental stages.
Specificity Index
To assess probe specificity, we compared the signal from oligonucleotides (probes) complementary to microRNAs (matched probe) and oligonucleotides with mismatches (mismatched probe, see above). Mismatched oligonucleotides were printed for the first 24 probes (EAM101, EAM103, . . . EAM147) and were named EAM102, EAM104, . . . EAM148. Mismatched oligonucleotides were spotted as nearest neighbors to microRNA oligonucleotides. To calculate the specificity index (FIG. 6A) we used datasets from two samples of each of the five time points from this study (5 time points×2 independent samples=10 hybridizations total). Calculations were based on cumulative signals from all experiments. EAM141, EAM143, EAM145 and EAM147 are let-7 family members and have very similar sequences. EAM117, EAM119 and EAM107 and EAM109 are also closely related. Therefore, there might be cross-reactivity within each of these groups. The matched/mismatched probe pair EAM135/EAM136 was excluded from FIG. 6A as EAM136 did not give a signal above background at any of the five time points.
Summary of Features on the Microarray (Probe Set)













Mouse microRNAs (Rfam 3.0)
129


Other mammalian microRNAs (Rfam 3.0, rat and human)
9


Other unique small RNAs
18


Total
156









Of the 156 unique small RNA probes, we found that 116 (74%) showed robust signals for at least one of the five time points. Of these, 83 changed significantly (ANOVA, P<0.001) and 66 changed by more than two-fold.

The predicted stem-loop RNA structures were generated using the mfold (version 3.1) software (Zuker Nucleic Acids Res, 31:3406-3415, 2003).
Northern Blots
Northern blots were performed as described (Lau et al., Science 294:858-862, 2001). 25 μg of total RNA was loaded per lane. A probe for the mouse U6 snRNA (5′-tgtgctgccgaagcgagcac-3′) was used as a loading control. The probe for each northern blots had the same sequences as the corresponding EAM# oligonucleotides printed on the microarray as shown in Table 2. Each blot was stripped by boiling for 5 minutes in distilled water and was reprobed up to four times. The probes used were as follows: EAM119 (miR-29b), EAM125 (miR-138), EAM224 (miR-17-5p), EAM234 (miR-199a), EAM131 (miR-92), EAM109 (miR-7), EAM150 (miR-9) and EAM103 (miR-124a).
Detectably Labeled microRNA
First, small RNAs (e.g., 18-26 nucleotides) are size-selected from total RNA, for example, by using denaturing polyacrylamide gel electrophoresis. Oligonucleotide linkers (e.g., DNA, RNA, RNA/DNA hybrid, or having a block at the 3′ end to inhibit self ligation) are attached to the 5′ and 3′ ends of the small RNAs. These linkers are at least 5, 10, 12, 15, 18, 20, or 25 nucleotides in length. Such linkers optionally include sites that facilitate subsequent cloning (e.g., restriction sites), sites that promote transcription (e.g., T7 site), or sites that facilitate the purification of the microRNA (e.g., a biotin).
Diagnostics
MicroRNAs or small noncoding RNAs are likely to be differentially expressed in a variety of pathologies. The methods of the invention are useful for the identification of microRNAs whose differential expression in associated with pathology. The identification of one or more microRNAs that are differentially expressed in a subject having a pathogy, relative to their expression in a normal control subject, indicates that the pathology is a microRNA-related condition, disease, or disorder.
A set of two or more differentially expressed microRNAs defines a microRNA expression profile. A specific microRNA expression profile may correlate with a particular disease state. Methods of the invention may be used for the analysis of a microRNA expression profile in a biological sample derived from a subject. The identification of a microRNA profile in a biological sample from a subject may indicate that the subject has a microRNA-related condition, disease, of disorder.
In one example, microRNAs are isolated from a neoplasm, and their expression is compared to the expression of microRNAs isolated from corresponding normal control tissue. One or more microRNAs that are differentially expressed in a neoplasm defines the microRNA expression profile of the neoplasm. The identification of an altered microRNA expression profile in a neoplastic tissues is useful in the diagnosis of a microRNA-related neoplasm. Such a neoplasm is treated with a therapeutic molecule that modulates microRNA expression. The treatment regimen is monitored by assaying for an alteration in the microRNA expression profile. A therapeutic molecule that normalizes the microRNA expression profile is useful in the methods of the invention.
Mutations in microRNAs and small noncoding RNAs, which normally function in hematopoietic development, are associated with cell cycle dysfunction and leukemia in humans. Similarly, mutations in the disclosed microRNAs and small noncoding RNAs (e.g., those listed in Table 1 or 2), which normally function in development, are likely to be associated with a microRNA-related disease, such as a neoplastic disease. The differential expression of at least one of the nucleic acids listed in Table 2 in a subject diagnosed as having a particular condition, disease, or disorder, relative to a normal control patient, indicates that the pathology is a microRNA-related condition, disease, or disorder.
In one example, oligonucleotides or longer fragments derived from any of the nucleic acid sequences described herein (e.g., those listed in Table 1 or Table 2) may be used as targets in a microarray. The microarray is used to assay the expression level of large numbers of microRNAs or small noncoding RNAs simultaneously and to identify genetic variants, mutations, and polymorphisms. Such information can be used to diagnose a microRNA-related condition, disease, or disorder.
In yet another example, hybridization with PCR probes that are capable of detecting at least one of the polynucleotide sequences listed in Table 1 or 2, including microRNA precursors, or closely related molecules, may be used to hybridize to a nucleic acid sequence derived from a patient having a microRNA-related condition, disease, or disorder. The specificity of the probe and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), determines whether the probe hybridizes to a naturally occurring sequence, allelic variants, or other related sequences. Hybridization techniques may be used to identify mutations indicative of a microRNA-related condition, disease, or disorder in a nucleic acid sequence listed in Table 1 or 2, or may be used to monitor expression levels of these nucleic acid molecules.
In yet another example, humans may be diagnosed for a propensity to develop a microRNA-related condition, disease, or disorder by direct analysis of the sequence of at least one of the nucleic acids listed in Table 1 or 2.
Microarrays
The nucleic acid molecules described herein (e.g., detectably labeled microRNAs that are amplified from a sample or that include a linker, or a nucleic acid molecule listed in Tables 1 or 2), or fragments thereof, are useful as hybridizable array elements in a microarray. The array elements are organized in an ordered fashion such that each element is present at a specified location on the substrate. Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, beads, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as expression levels of particular genes or proteins.
Alternatively, an array element is identified not by its geographical location, but because it is linked to an identifiable substrate. The substrate would necessarily have a characteristic (e.g., size, color, fluorescent label, charge, or any other identifiable signal) that allows the substrate and its linked nucleic acid molecule to be distinguished from other substrates with linked nucleic acid molecules. The association of the array element with an identifiable substrate allows hybridization patterns and intensities to be interpreted as expression levels of particular genes. In one example, a nucleic acid molecule is affixed to a bead that fluoresces at a particular wave length. Binding of a detectably labeled microRNA to the oligonucleotide alters the fluorescence of the bead. Such binding can be detected using standard methods.
Methods for making nucleic acid microarrays are known to the skilled artisan and are described, for example, in U.S. Pat. No. 5,837,832, Lockhart, et al. (Nat Biotech 14:1675-1680, 1996), and Schena, et al. (Proc Natl Acad Sci USA 93:10614-10619, 1996), herein incorporated by reference. Methods for making polypeptide microarrays are described, for example, by Ge (Nucleic Acids Res 28:e3.i-e3.vii, 2000), MacBeath et al., (Science 289:1760-1763, 2000), Zhu et al. (Nat Genet 26:283-289), and in U.S. Pat. No. 6,436,665, hereby incorporated by reference.
Nucleic Acid Microarrays
To produce a nucleic acid microarray oligonucleotides may be synthesized or bound to the surface of a substrate using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.), incorporated herein by reference. Alternatively, a gridded array may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedure.
A nucleic acid molecule (e.g., RNA or DNA) derived from a biological sample may be used to produce a hybridization probe as described herein. The biological samples are generally derived from a patient, preferably as a bodily fluid (such as blood, cerebrospinal fluid, phlegm, saliva, or urine) or tissue sample (e.g., a tissue sample obtained by biopsy). For some applications, cultured cells (e.g., lymphocytes) or other tissue preparations may be used. The mRNA is isolated according to standard methods, and cDNA is produced and used as a template to make complementary RNA suitable for hybridization. Such methods are described herein. The RNA is amplified in the presence of fluorescent nucleotides, and the labeled probes are then incubated with the microarray to allow the probe sequence to hybridize to complementary oligonucleotides bound to the microarray. Such hybridization methods are described herein. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously (e.g., Heller et al., Proc Natl Acad Sci USA 94:2150-2155, 1997). Preferably, a scanner is used to determine the levels and patterns of fluorescence.
Screening Assays
The invention described herein also provides screening methods for the identification of therapeutic molecules for the treatment or prevention of a microRNA-related condition, disease, or disorder.
In addition, the microRNA sequences described herein are useful as therapeutic targets for the treatment of a microRNA-related pathology. These compositions of the invention are useful for the high-throughput, low-cost screening of candidate compounds to identify those that modulate the expression of a microRNA whose expression is altered in a patient having a microRNA-related condition, disease, or disorder. In one embodiment, the effects of known therapeutic drugs on the expression of a microRNA can be assayed using the methods of the invention. Tissues or cells treated with these drugs are compared to untreated corresponding control samples to produce expression profiles of known therapeutic agents. Knowing the identity of sequences that are differentially regulated in the presence and absence of a therapeutic agent is useful in understanding the mechanisms of drug action.
Any number of methods are available for carrying out screening assays to identify new candidate compounds that modulates the expression of a microRNA. In one working example, candidate compounds are added at varying concentrations to the culture medium of cultured cells expressing one of the nucleic acid sequences of the invention. MicroRNA expression is then measured, for example, by microRNA microarray analysis, Northern blot analysis (Ausubel et al., supra), reverse transcriptase PCR, or quantitative real-time PCR using any appropriate fragment prepared from the nucleic acid molecule as a hybridization probe. The level of gene expression in the presence of the candidate compound is compared to the level measured in a control culture medium lacking the candidate molecule. A compound that modulates the expression of a microRNA, or a functional equivalent thereof, is considered useful in the invention; such a molecule may be used, for example, as a therapeutic to treat a microRNA-related condition, disease, or disorder in a human patient.
In yet another working example, candidate compounds may be screened for those that specifically bind to a microRNA (e.g., a microRNA listed in Table 1 or 2) or a microRNA precursor. The efficacy of a candidate compound is dependent upon its ability to interact with such a microRNA or with a microRNA precursor. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays (e.g., those described in Ausubel et al., supra). In one embodiment, a candidate compound may be tested in vitro for its ability to specifically bind a microRNA or a microRNA precursor of the invention. In another embodiment, a candidate compound is tested for its ability to enhance the biological activity of a microRNA or a microRNA precursor described herein. The biological activity of a microRNA or a microRNA precursor may be assayed using any standard method, for example, by assaying the expression of a genetic target of the microRNA.
In another working example, a nucleic acid described herein (e.g., a nucleic acid listed in Table 1 or 2) is expressed as a transcriptional or translational fusion with a detectable reporter, and expressed in an isolated cell (e.g., mammalian or insect cell) under the control of a promoter, such as the microRNA's own promoter or a heterologous promoter, such as an inducible promoter. The cell expressing the fusion protein is then contacted with a candidate compound, and the expression of the detectable reporter in that cell is compared to the expression of the detectable reporter in an untreated control cell. A candidate compound that alters (e.g., increases or decreases) the expression of the detectable reporter is a compound that is useful for the treatment of a microRNA-related disease or disorder.
Candidate compounds include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acids, and antibodies that bind to a nucleic acid sequence of the invention (e.g., those listed in Table 1 or 2). For those nucleic acid sequences or polypeptides whose expression is decreased in a patient having a microRNA-related condition, disease, or disorder, agonists would be particularly useful in the methods of the invention. For those nucleic acid molecules or polypeptides whose expression is increased in a patient having a microRNA-related condition, disease, or disorder, antagonists would be particularly useful in the methods of the invention.
Small molecules of the invention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules.
Test Compounds and Extracts
In general, compounds capable of altering the expression or activity of a microRNA are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries or from polypeptide or nucleic acid libraries (e.g., a library containing nucleic acid molecules listed in Table 1 or 2), according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Compounds used in screens may include known compounds (for example, known therapeutics used for other diseases or disorders). Alternatively, virtually any number of unknown chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.
In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their molt-disrupting activity should be employed whenever possible.
When a crude extract is found to alter the expression or activity of a microRNA, or found to bind a microRNA, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that alters the expression or activity of a microRNA, or that binds a microRNA. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful as therapeutics for the treatment of a microRNA related disorder are chemically modified according to methods known in the art.
OTHER EMBODIMENTS
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication was specifically and individually indicated to be incorporated by reference.

## Claims

1. A method for identifying microRNA expression in a sample, said method comprising:
(a) providing RNA from said sample, wherein said RNA comprises a microRNA;
(b) appending at least two linkers to said microRNA;
(c) detectably labeling said microRNA of step (b);
(d) contacting a microarray comprising at least 2 oligonucleotides with said detectably labeled microRNA; and
(e) detecting binding of said detectably labeled microRNA to said microarray.

2. The method of claim 1, wherein said RNA is isolated microRNA.

3. The method of claim 1, wherein said linker comprises an oligonucleotide.

4. The method of claim 3, wherein said linker is an RNA/DNA hybrid.

5. The method of claim 1, wherein said linker is appended in a ligation reaction.

6. The method of claim 1, wherein said microRNA is useful as a template for a reverse transcriptase polymerase chain reaction (RT-PCR).

7. The method of claim 1, wherein said microRNA is detectably labeled during the performance of the polymerase chain reaction (PCR).

8. The method of claim 1, wherein at least one of said linkers comprises at least one restriction site.

9. The method of claim 1, wherein said sample is a tissue sample.

10. A method for identifying microRNA expression, said method comprising:
(a) providing a microRNA isolated from a sample;
(b) amplifying said microRNA to produce a detectably labeled microRNA;
(c) contacting a microarray comprising at least 2 oligonucleotides with said detectably labeled microRNA; and
(d) detecting binding of said detectably labeled microRNA to said microarray.

11. The method of claim 10, wherein said microRNA is detectably labeled during the performance of PCR.

12. The method of claim 10, wherein said detectable label is a fluorophore.

13. The method of claim 10, wherein said detectable label is detected by analyzing enzyme activity, by direct immunoassay, or by a radiometric assay.

14. The method of claim 10, wherein said sample is a tissue sample.

15. The method of claim 1 or 10, wherein said sample is a neoplastic tissue sample.

16. The method of claim 1 or 10, wherein said microarray comprises at least 10 oligonucleotides.

17. The method of claim 16, wherein said microarray comprises at least 25 oligonucleotides.

18. The method of claim 17, wherein said microarray comprises at least 50 oligonucleotides.

19. The method of claim 18, wherein said microarray comprises at least 100 oligonucleotides.

20. A method for identifying microRNA expression in a sample, said method comprising:
(a) providing RNA from said sample, wherein said RNA comprises a microRNA;
(b) appending at least one linker to said microRNA, wherein said linker comprises a T7 promoter;
(c) detectably labeling said microRNA of step (b);
(d) contacting a microarray comprising at least 2 oligonucleotides with said detectably labeled microRNA; and
(e) detecting binding of said detectably labeled microRNA to said microarray.

21. A method for identifying differential expression of a microRNA in a test sample, said method comprising:
(a) providing a microRNA isolated from a test sample;
(b) amplifying said microRNA to produce a detectably labeled microRNA;
(c) contacting a microarray comprising at least 2 oligonucleotides with said detectably labeled microRNA; and
(d) detecting differential binding of said detectably labeled microRNA to said microarray, relative to the binding of a corresponding detectably labeled microRNA isolated from a control.

22. The method of claim 21, wherein said test sample is a tissue sample from a subject having a disease, condition, or disorder selected from the group consisting of autoinflammatory disorders, proliferative diseases, cardiovascular diseases, obesity, or an obesity related diseases.

23. The method of claim 22, wherein said proliferative disorder is selected from the group consisting of leukemias, lymphomas, sarcomas and carcinomas.

24. The method of claim 22, wherein said autoinflammatory disorder is selected from the group consisting of asthma, allergic intraocular inflammatory diseases, arthritis, atopic dermatitis, atopic eczema, diabetes, haemolytic anaemia, inflammatory dermatoses, inflammatory bowel or gastrointestinal disorders, multiple sclerosis, myasthenia gravis, pruritis/inflammation, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

25. The method of claim 22, wherein said cardiovascular disease is atherosclerosis, hypertension, cardiac artery disease, myocardial infarction, or congestive heart failure.

26. The method of claim 22, wherein said obesity-related disease is diabetes.

27. A method for producing a microRNA microarray, said method comprising:
(a) providing a microRNA;
(b) amplifying said microRNA; and
(c) affixing said microRNA to a solid support.

28. A method for identifying microRNA expression in a cell, said method comprising:
(a) amplifying a microRNA from a sample;
(b) detectably labeling said microRNA;
(c) contacting a microarray with said labeled microRNA; and
(d) measuring binding of said labeled microRNA to said microarray, wherein binding identifies said microRNA as being a microRNA expressed in said cell.

29. The method of claim 28, wherein said detectable label is a fluorophore.

30. The method of claim 29, wherein said binding alters the fluorescence of said fluorophore.

31. A method for identifying microRNA expression in a sample, said method comprising:
(a) providing RNA from said sample, wherein said RNA comprises a microRNA;
(b) appending at least one linker to said microRNA;
(c) detectably labeling said microRNA of step (b) during the performance of PCR;
(d) contacting a microarray comprising at least 2 oligonucleotides with said detectably labeled microRNA; and
(e) detecting binding of said detectably labeled microRNA to said microarray.

32. A method of labeling a microRNA, said method comprising:
(a) appending at least one linker to said microRNA;
(b) detectably labeling said microRNA of step (b) during the performance of PCR; thereby labeling said microRNA.

33. The method of claim 32, wherein said linker is an RNA/DNA hybrid.

34. The method of claim 32, wherein reverse transcription of said microRNA is performed prior to step (b).

35. The method of claim 32, wherein step (b) includes appending at least two linkers to said microRNA.

36. A method for identifying microRNA expression, said method comprising:
(a) attaching to a microRNA a linker bound to a detectable label;
(b) contacting a microarray comprising at least 2 oligonucleotides with said detectably labeled microRNA; and
(c) detecting binding of said detectably labeled microRNA to said microarray.

37. The method of claim 36, wherein said microRNA is isolated microRNA.

38. The method of claim 36, wherein said linker comprises an oligonucleotide.

39. The method of claim 36, wherein said detectable label is a fluorophore.

40. The method of claim 36, wherein said detectable label is detected by analyzing enzyme activity, by direct inimunoassay, or by a radiometric assay.

41. The method of claim 36, wherein said microRNA is from a tissue sample.

42. The method of claim 36, wherein said microarray comprises at least 10 oligonucleotides.

43. The method of claim 42, wherein said microarray comprises at least 50 oligonucleotides.

44. A method for identifying microRNA expression in a sample, said method comprising:
(a) providing RNA from said sample, wherein said RNA comprises a microRNA;
(b) appending at least one linker to a microRNA;
(c) detectably labeling said microRNA of step (b);
(d) contacting a microarray comprising at least 2 oligonucleotides with said detectably labeled microRNA; and
(e) detecting binding of said detectably labeled microRNA to said microarray.

45. The method of claim 44, wherein said RNA is isolated microRNA.

46. The method of claim 44, wherein said linker comprises an oligonucleotide.

47. The method of claim 46, wherein said linker is an RNA/DNA hybrid.

48. The method of claim 44, wherein said linker is appended in a ligation reaction.

49. The method of claim 44, wherein said sample is a tissue sample.

50. The method of claim 44, wherein said microarray comprises at least 10 oligonucleotides.

51. The method of claim 50, wherein said microarray comprises at least 50 oligonucleotides.