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Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing.

Methods (San Diego, Calif.) (2007-12-26)
Markus Hafner, Pablo Landgraf, Janos Ludwig, Amanda Rice, Tolulope Ojo, Carolina Lin, Daniel Holoch, Cindy Lim, Thomas Tuschl
RESUMO

Distinct classes of small RNAs, 20-32 nucleotides long, play important regulatory roles for diverse cellular processes. It is therefore important to identify and quantify small RNAs as a function of development, tissue and cell type, in normal and disease states. Here we describe methods to prepare cDNA libraries from pools of small RNAs isolated from organisms, tissues or cells. These methods enable the identification of new members or new classes of small RNAs, and they are also suitable to obtain miRNA expression profiles based on clone count frequencies. This protocol includes the use of new deep sequencing methods (454/Roche and Solexa) to facilitate the characterization of diverse sequence pools of small RNAs.

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Sigma-Aldrich
Adenosine 5′-monophosphate monohydrate, from yeast, ≥97%
Sigma-Aldrich
Aldrithiol-2, 98%
Sigma-Aldrich
Albumin, Acetylated from bovine serum, protease free, for molecular biology