Thursday, February 10, 2011

Circligase II-based detection of miRNAs

In a recent publication, Kumar et al. demonstrate the power of their “miR-ID” method in expression profiling of microRNA (miRNA) molecules. The authors used a ligase-based recircularization procedure and compared T4 RNA Ligase to Epicentre’s Circligase™ II enzyme (typically used for ssDNA circularization) in recircularization of miRNAs. They found that, while both ligases were able to join the ends of standard miRNA molecules with 2’ and 3’-hydroxyl groups, only Circligase II-based reactions were able to ligate the ends of miRNAs that contained 2’-O-methyl groups (common in plant miRNAs). Following recircularization, the miRNAs were reverse-transcribed to form tandem repeats of the sequence that is complementary to the miRNA, and then amplified by qPCR to develop an expression profile of miRNAs in a given sample.

The authors state that no chemically modified probes or primers are required for their method. The key elements of the miR-ID process (miRNA circularization, reverse transcription of circularized RNA, and qPCR using 5’-overlapping primers) are likely to be of general interest in other transcriptome discovery techniques.

ResearchBlogging.orgKumar, P. et al. (2010). miR-ID: A novel, circularization-based platform for detection of microRNAs RNA, 17 (2), 365-380 DOI: 10.1261/rna.2490111

No comments: