Identification of microRNAs and Other Tiny Noncoding RNAs by cDNA Cloning
- 1.6k Downloads
MicroRNAs (miRNAs) and other small RNAs can be identified by cloning and sequencing cDNAs prepared from the ∼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 λ 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.
Key WordsmicroRNA noncoding RNA RNA ligase SMART RNA folding cDNAs Northern blot
- 21.Pfeffer, S., Lagos-Quintana, M., and Tuschl, T. (2003) Cloning of small RNA molecules, in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Chanda, V. B., Kingston, R. E., Moore, D. D., Seidmann, J. G., Smith, J. A., and Struhl, K., eds.), Wiley and Sons, New York.Google Scholar