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Moreover, the present invention concerns methods and compositions for generating miRNA profiles and employing such profiles for therapeutic, diagnostic, and prognostic applications. Field of the Invention The present invention relates generally to the field of molecular biology.

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Methods and compositions for isolating, labeling, preparing miRNAs for analysis or ACTION ACTINA NEVADA 700X a tool for analysis are described, such as miRNA arrays. In addition, there are applications for miRNAs in diagnostics, therapeutics, and prognostics. Several hundreds of miRNAs have been identified in plants and animals — including humans — which do not appear to have endogenous siRNAs. See review of Carrington et al The precursors form structures that fold back on each other in self- complementary regions; they are then processed by the nuclease Dicer in animals or DCL1 in plants.

The function of most miRNAs is not known. A number of miRNAs, however, seem to be involved in gene regulation. Some miRNAs, such as lin-4, let-7, mir, mir, and bantam, have been shown to play critical roles in cell differentiation and tissue development Ambros, ; Xu et al, Others are believed to have similarly important roles because of their differential spatial and temporal expression ACTION ACTINA NEVADA 700X.

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Research on microRNAs miRNAs is increasing as scientists are beginning to appreciate the broad role that these molecules play in the regulation of eukaryotic gene expression. The two ACTION ACTINA NEVADA 700X understood miRNAs, lin-4 and let-7, regulate developmental timing in C.

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Several hundred miRNAs have been identified in C. As would be expected for molecules that regulate gene expression, miRNA levels have been ACTION ACTINA NEVADA 700X to vary between tissues and developmental states. Characterization of a number of miRNAs indicates that they influence a variety of processes, including early development Reinhart, 'cell proliferation and cell death Brennecke,and apoptosis and fat metabolism Xu, In addition, one study shows a strong correlation between reduced expression of two miRNAs and chronic lymphocytic leukemia, providing a possible link between miRNAs and cancer Calin, Although the field is still young, there is speculation that miRNAs could be as important as transcription factors in regulating gene expression in higher eukaryotes.

Several publications describe labeling miRNAs for analysis. These publications describe appending a radioactive phosphate at the 5' end of the miRNA population using a polynucleotide kinase Krichevsky, or a radiolabeled, single nucleotide at the 3' end with RNA ligase Dostie, For the purpose of using arrays to estimate the relative abundances of miRNAs in samples, these methods have two significant drawbacks: Furthermore, while RNA oligonucleotides have been labeled with non-isotopic labels Martin et al,there is no evidence that small RNA molecules from a cell ACTION ACTINA NEVADA 700X can be effectively labeled in a similar manner after they have been enriched or isolated from the lysate.

Because microarrays are typically used to analyze messenger RNAs that are hundreds or thousands of nucleotides in length, we found that the mer probes typically used in microarrays were not compatible with miRNA analysis.

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Therefore, there is a need for information about the function and ACTION ACTINA NEVADA 700X of miRNAs, as well as for methods and compositions that can be used for their characterization and analysis. The term "miRNA" is used according to its ordinary and plain meaning and refers to a microRNA molecule found in eukaryotes that is involved in RNA-based gene regulation. See, e.

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The term will be used to refer to the RNA molecule processed from a precursor. Individual miRNAs have been identified and sequenced in different organisms, and they have been given names.

Names of miRNAs and their sequences are provided herein. Additionally, other miRNAs are known to those of skill in the art and can be readily implemented in embodiments of the invention. The methods and compositions should not be limited to miRNAs identified ACTION ACTINA NEVADA 700X the application, as they are provided as examples, not necessarily as limitations of the invention. Some embodiments ot trie invention concern methods for labeling miRNA.

While it is contemplated that miRNA may be labeled with a single label, in many embodiments of the invention, multiple labels may be the same or different labels are attached to the miRNA. Consequently, methods for multi-labeling are specifically contemplated as part of the invention. In ACTION ACTINA NEVADA 700X embodiments, the miRNA to be labeled is contacted with an enzyme that ACTION ACTINA NEVADA 700X the addition of di- or tri-phosphate nucleotides to the 3' end of the miRNA, effectively adding a nucleotide tail to the miRNA. The miRNA and enzyme will be incubated under conditions to allow catalysis by the enzyme to occur such that a reaction mixture for tailing is formed.

Moreover, the reaction mixture will contain nucleotides that are labeled or unlabeled, which are added to the 3' end ofthe miRNA by the enzyme to form a tailed miRNA molecule. The term "nucleotide" refers to both ribonucleotides and deoxyribonucleotides, though it is specifically contemplated that any embodiment discussed in the context of "nucleotides" may be implemented specifically with "ribonucleotides" or "deoxyribonucleotides," unless otherwise specified or limited.

In specific embodiments, the nucleotide is uridine, which may or ACTION ACTINA NEVADA 700X not be modified as discussed below. The tail added to the miRNA is at least or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81? The nucleotides added to the miRNA may already have a label attached or the label may be attached after the miRNA is tailed. Download the latest drivers for your Action ACTINA NEVADA X to keep your Computer up-to-date.

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