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Open Access Research article

Reliable reference miRNAs for quantitative gene expression analysis of stress responses in Caenorhabditis elegans

Konstantinos Kagias, Agnieszka Podolska and Roger Pocock

Author Affiliations

Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen, Denmark

BMC Genomics 2014, 15:222  doi:10.1186/1471-2164-15-222

Published: 21 March 2014

Abstract

Background

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.

Results

Here we 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. We conducted genome-wide RNA-seq for small RNAs and selected abundant miRNAs with minimal variation of expression between the different conditions. We 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, we were able to successfully identify suitable reference miRNAs for each stress condition. Interestingly, we 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.

Conclusions

Our 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.

Keywords:
Reference genes; miRNA; qPCR analysis; Normalization