Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies

Giacomo Spinsanti,Cristina Panti,Francesco Frati,Silvia Casini,Elisa Lazzeri,Cristina Maria Fossi,Letizia Marsili

doi:10.1186/1471-2199-7-32

Abstract

BackgroundOdontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR) has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers.ResultsTen commonly used housekeeping genes (HKGs) were partially sequenced in the striped dolphin (Stenella coeruleoalba) and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper) which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH) and tyrosine 3-monooxygenase (YWHAZ) always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4) and S18 (RPS18) also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC), phosphoglycerate kinase 1 (PGK1), hypoxanthine ribosyltransferase (HPRT1) and β-2-microglobin (B2M) show variable expression among the studied samples and appear as less suitable reference genes for data normalization.ConclusionIn this work, we have provided essential background information for the selection of control genes in qRT-PCR studies of cetacean skin biopsies, as a molecular technique to investigate ecotoxicological hazard in marine mammals. Of 10 HKGs tested, those encoding for YWHAZ and GAPDH appear as the most reliable control genes for the normalization of qRT-PCR data in the analysis of striped dolphin skin biopsies. Potentially useful reference genes are also those encoding for ribosomal proteins L4 and S18.

Highlights

Odontocete cetaceans occupy the top position of the marine food-web and are sensitive to the bioaccumulation of lipophilic contaminants
In this work, we have provided essential background information for the selection of control genes in quantitative real-time PCR (qRT-PCR) studies of cetacean skin biopsies, as a molecular technique to investigate ecotoxicological hazard in marine mammals
Expression patterns of the ten house-keeping genes (HKGs) have been subsequently evaluated in skin biopsies (n = 30) obtained using non-lethal techniques, in a Mediterranean sub-population of the striped dolphin from the Ligurian Sea

Summary

Introduction

Odontocete cetaceans occupy the top position of the marine food-web and are sensitive to the bioaccumulation of lipophilic contaminants. Quantitative real-time PCR (qRT-PCR) represents a rapid and reliable method for the detection and quantification of mRNA transcription levels of a selected gene of interest (GOI). QRT-PCR may be effectively used to evaluate the levels of expression of biomarker genes under the induction of xenobiotic contaminants. Among several strategies proposed [2,3], house-keeping genes (HKGs) are commonly accepted and frequently used to normalize qRT-PCR and to reduce possible errors generated in the quantification of gene expression. In this normalization strategy, internal controls are subjected to the same conditions as the RNA of interest and their expression measured by qRT-PCR. With the introduction of qRT-PCR, other ordinarily used HKGs, involved with basic and ubiquitous cellular functions and belonging to different functional classes, have been introduced [21,22,23,24,25]

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Conclusion