title: An efficient and sensitive method for preparing cDNA libraries from scarce biological samples.
abstract: The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids.
authors: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors: Catherine H
authors: CH
authors: Sterling
authors: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors: Isana
authors: I
authors: Veksler-Lublinsky
authors: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA victor.ambros@umassmed.edu.
authors: Victor
authors: V
authors: Ambros
mesh_terms: D019921:Biotinylation; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D003856:Deoxyuracil Nucleotides; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D035683:MicroRNAs / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D059368:RNA Isoforms / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D018076:DNA, Complementary; D003856:Deoxyuracil Nucleotides; D035683:MicroRNAs; D059368:RNA Isoforms