Stem‐Loop RT‐qPCR for miRNAs

Abstract This unit presents a specific and sensitive quantitative reverse‐transcription PCR (RT‐qPCR) method for measuring individual microRNAs (miRNAs) in tissue or cultured cells. miRNAs are 17 to 24 nucleotides (nt) in length. Standard and quantitative PCR methods require a template that is at least two times the length of either of the specific forward or reverse primers, each typically ∼20 nt in length. Thus, the target minimum length is ≥40 nt, making miRNAs too short for standard RT‐qPCR methods. In this assay, each of the RT‐qPCR nucleic acid reagents, including the RT‐primer, the forward and reverse PCR primers, and the hydrolysis probe, contain design features that, together, optimize miRNA specificity and assay sensitivity. The RT‐primer contains a highly stable stem‐loop structure that lengthens the target cDNA. The forward PCR primer adds additional length with nucleotides that optimize its melting temperature ( T m ) and enhance assay specificity. The reverse primer disrupts the stem loop. Assay specificity is further optimized by placement of the probe over much of the original miRNA sequence, and the probe T m is optimized by addition of a minor groove binding (MGB) moiety. Curr. Protoc. Mol. Biol . 95:15.10.1‐15.10.15. © 2011 by John Wiley & Sons, Inc.