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

• Published in: Methods (San Diego, Calif.) Vol. 44; no. 1; pp. 3 - 12
• Main Authors: Hafner, Markus, Landgraf, Pablo, Ludwig, Janos, Rice, Amanda, Ojo, Tolulope, Lin, Carolina, Holoch, Daniel, Lim, Cindy, Tuschl, Thomas
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 2008
• Subjects: Animals; cDNA libraries; Deep sequencing; Gene Library; Humans; MicroRNAs - genetics; MicroRNAs - isolation & purification; miRNA; Oligonucleotide Array Sequence Analysis - methods; Regulatory Sequences, Ribonucleic Acid - genetics; Small RNA; Small RNA libraries; Small RNA libraries; Deep sequencing; miRNA; cDNA libraries; Small RNA
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2007.09.009

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.