Selection of suitable reference genes for gene expression analysis in gills and liver of fish under field pollution conditions

Noemí Rojas-Hernandez,Caren Vega-Retter,David Véliz

doi:10.1038/s41598-019-40196-3

Abstract

To understand the role of gene expression in adaptive variation, it is necessary to examine expression variation in an ecological context. Quantitative real-time PCR (qPCR) is considered the most accurate and reliable technique to measure gene expression and to validate the data obtained by RNA-seq; however, accurate normalization is crucial. In Chile, the freshwater silverside fish Basilichthys microlepidotus inhabits both polluted and nonpolluted areas, showing differential gene expression related to pollution. In this study, we infer the stability of six potential reference genes (tubulin alpha, hypoxanthine-guanine phosphoribosyltransferase, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, 60S ribosomal protein L13, and 60S ribosomal protein L8) in the gills and liver of silverside individuals inhabiting polluted and nonpolluted areas. To validate the reference genes selected, the most and least stable reference genes were used to normalize two target transcripts, one for each organ. The RefFinder tool was used to analyze and identify the most stably expressed genes. The 60S ribosomal protein L8 gene was ranked as the most stable gene for both organs. Our results show that reference gene selection influences the detection of differences in the expression levels of target genes in different organs and, also highlighting candidate reference genes that could be used in field studies.

Highlights

Water pollution is one of the main threats to global freshwater biodiversity that, together with other human interventions, has impacted freshwater species worldwide[1,2]
Whitehead and Crawford argued for the necessity of examining expression variation in ecological contexts to understand the role of gene expression in adaptive variation[6]
Quantitative real-time PCR is considered the most accurate and reliable technique to measure gene expression and to validate the data obtained by microarrays and RNA-Seq[11]

Summary

Introduction

Water pollution is one of the main threats to global freshwater biodiversity that, together with other human interventions, has impacted freshwater species worldwide[1,2]. Vega-Retter et al described candidate loci under directional selection related to the pollution[21] and found differential gene expression when comparing individuals inhabiting polluted and nonpolluted areas, with the differential gene expression relating to apoptotic processes and cellular proliferation as well as the suppression and progression of tumors[22]. This system allows for the description of genes with stable expression profiles in a complex and dynamic environment where multiple and simultaneous stimuli occur, providing insights into genes that can be used as reference genes in ecological contexts. The map3k5 gene is involved in metal ion binding and in biological processes, such as innate immune response, positive regulation of apoptotic process, cellular response to tumor necrosis factor and positive regulation of transcription, DNA-templated

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