Identification of microRNAs and other tiny noncoding RNAs by cDNA cloning.

MicroRNAs (miRNAs) and other small RNAs can be identified by cloning and sequencing cDNAs prepared from the approximately 22-nt fraction of total RNA. Methods are described for the construction of cDNA libraries from small noncoding RNAs through the use of T4 RNA ligase, reverse transcriptase, and polymerase chain reaction. cDNAs are cloned in lambda or plasmid vectors, and the sequences are compared to annotated genomic sequence databases, and analyzed by RNA folding programs to distinguish miRNA sequences from other small RNAs of similar size. Northern blot hybridization is used to confirm the expression of small RNAs in vivo.

[1]  C. Burge,et al.  The microRNAs of Caenorhabditis elegans. , 2003, Genes & development.

[2]  T. Tuschl,et al.  New microRNAs from mouse and human. , 2003, RNA.

[3]  J. Sabina,et al.  Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. , 1999, Journal of molecular biology.

[4]  M. A. Rector,et al.  Endogenous and Silencing-Associated Small RNAs in Plants Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.003210. , 2002, The Plant Cell Online.

[5]  L. Lim,et al.  An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans , 2001, Science.

[6]  S. Eddy.  Computational Genomics of Noncoding RNA Genes , 2002, Cell.

[7]  V. Ambros.  microRNAs Tiny Regulators with Great Potential , 2001, Cell.

[8]  B. Reinhart,et al.  The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans , 2000, Nature.

[9]  G. Ruvkun,et al.  A uniform system for microRNA annotation. , 2003, RNA.

[10]  V. Ambros,et al.  An Extensive Class of Small RNAs in Caenorhabditis elegans , 2001, Science.

[11]  C. Llave,et al.  Cleavage of Scarecrow-like mRNA Targets Directed by a Class of Arabidopsis miRNA , 2002, Science.

[12]  T. Tuschl,et al.  Identification of Novel Genes Coding for Small Expressed RNAs , 2001, Science.

[13]  T. Tuschl,et al.  Identification of Tissue-Specific MicroRNAs from Mouse , 2002, Current Biology.

[14]  C. Burge,et al.  Vertebrate MicroRNA Genes , 2003, Science.

[15]  V. Ambros,et al.  The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 , 1993, Cell.

[16]  L. Orgel,et al.  Preparation of ligation intermediates and related polynucleotide pyrophosphates. , 1984, Biochimica et biophysica acta.

[17]  E. Moss,et al.  Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation. , 2002, Developmental biology.

[18]  T. Tuschl,et al.  RNA interference is mediated by 21- and 22-nucleotide RNAs. , 2001, Genes & development.