There is no magic bullet: the importance of testing reference gene stability in RT-qPCR experiments across multiple closely related species.

Bert Foquet,Hojun Song

doi:10.7717/peerj.9618

Abstract

Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) is the current gold standard tool for the study of gene expression. This technique is highly dependent on the validation of reference genes, which exhibit stable expression levels among experimental conditions. Often, reference genes are assumed to be stable a priori without a rigorous test of gene stability. However, such an oversight can easily lead to misinterpreting expression levels of target genes if the references genes are in fact not stable across experimental conditions. Even though most gene expression studies focus on just one species, comparative studies of gene expression among closely related species can be very informative from an evolutionary perspective. In our study, we have attempted to find stable reference genes for four closely related species of grasshoppers (Orthoptera: Acrididae) that together exhibit a spectrum of density-dependent phenotypic plasticity. Gene stability was assessed for eight reference genes in two tissues, two experimental conditions and all four species. We observed clear differences in the stability ranking of these reference genes, both between tissues and between species. Additionally, the choice of reference genes clearly influenced the results of a gene expression experiment. We offer suggestions for the use of reference genes in further studies using these four species, which should be taken as a cautionary tale for future studies involving RT-qPCR in a comparative framework.

Highlights

The current gold standard tool for studying gene expression at the RNA level is Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR or qPCR), due to its high sensitivity and speed of analysis (Gachon, Mingam & Charrier, 2004; Thellin et al, 2009)
We explore the merit of this assumption by testing reference gene stability in qPCR experiments in four closely related species of grasshoppers in the genus Schistocerca (Orthoptera: Acrididae)
A total of 9 potential reference genes were initially selected based on previous studies using qPCR for locust gene expression research (Chapuis et al, 2011; Van Hiel et al, 2009; Yang et al, 2014)

Summary

Introduction

The current gold standard tool for studying gene expression at the RNA level is Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR or qPCR), due to its high sensitivity and speed of analysis (Gachon, Mingam & Charrier, 2004; Thellin et al, 2009). It is often the case that certain reference genes are selected for a particular qPCR experiment because they have been used previously, either for other experimental conditions or even in other tissues and species This type of blind adoption of reference genes can result in inaccurate normalization of target gene expression, and in an incorrect interpretation of the results (Bustin et al, 2013; Dheda et al, 2004; Gutierrez et al, 2008; Nicot et al, 2005; Tricarico et al, 2002; Vandesompele et al, 2002). It is critical that a thorough investigation of reference gene stability is needed prior to setting up any qPCR experiment

Objectives

Methods

Results

Discussion

Conclusion