qPCR multiplex detection of microRNA and messenger RNA in a single reaction

Abstract

Reverse Transcription-Quantitative PCR (RT-qPCR) is one of the standards for analytical measurement of different RNA species in biological models. However, current Reverse Transcription (RT) based priming strategies are unable to synthesize differing RNAs and ncRNAs especially miRNAs, within a single tube. We present a new methodology, referred to as RNAmp, that measures in parallel miRNA and mRNA expression. We demonstrate this in various cell lines, then evaluate clinical utility by quantifying several miRNAs and mRNA simultaneously in sera. PCR efficiency in RNAmp was estimated between 1.8 and 1.9 which is comparable to standard miRNA and random primer RT approaches. Furthermore, when using RNAmp to detect selected mRNA and miRNAs, the quantification cycle (Cq) was several cycles lower. This low volume single-tube duplex protocol reduces technical variation and reagent usage and is suitable for uniform analysis of single or multiple miRNAs and/or mRNAs within a single qPCR reaction.