Reliable reference miRNAs for quantitative gene expression analysis identified by RNA-Seq

Quantitative real-time PCR (qPCR) has become the “gold standard”for measuring expression levels of individual miRNAs. However, little is known about the validity of reference miRNAs, the improper use of which can result in misleading interpretation of data.

Here researchers from the University of Copenhagen undertook a systematic approach to identify highly stable miRNAs in different stress conditions such as low oxygen (hypoxia), UV-stress and high temperature (heat-stress) in the nematode Caenorhabditis elegans.

They conducted genome-wide RNA-seq for small RNAs and selected abundant miRNAs with minimal variation of expression between the different conditions. They further validated the stable expression of a selection of those constitutively expressed candidates in the different stress conditions by SYBR Green qPCR.

The selected miRNA candidates were analyzed for stability by applying the widely used geNorm logarithm. With this approach, the researchers were able to successfully identify suitable reference miRNAs for each stress condition. Interestingly, they also found that 3 miRNAs, namely mir-2-5p, mir-46-3p and mir-47-3p, are stable in all the above-mentioned conditions suggesting that they might have general functions independent of stress.

This analysis offers a comprehensive list of stably expressed miRNAs in different stress conditions that can be confidently used as reference miRNAs for qPCR analysis in C. elegans.