Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas).

I-Hua Chen,Jiann-Hsiung Wang,Wen-Been Chang,Tsung-Hsien Li,Wei Cheng Yang,Yeong-Huey Wu,Shih-Jen Chou,Ming-Yih Leu

doi:10.7717/peerj.1810

Abstract

Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults.

Highlights

Reverse transcription quantitative PCR (RT-qPCR) is considered the ideal method in gene expression studies because of its high sensitivity, time efficiency, and reliability (Derveaux, Vandesompele & Hellemans, 2010; Pfister, Tatabiga & Roser, 2011)
The four algorithms used to assess the stability of housekeeping genes (HKGs), geNorm, NormFinder, BestKeeper, and comparative Ct represent feasible strategies, none of them are currently considered to be the best
BestKeeper uses raw Cq data instead of the relative expression level employed by geNorm and NormFinder for selecting the least variable gene, and it has been shown that this may lead to the different outputs among these three methods (Scharlaken et al, 2008)

Summary

Introduction

Reverse transcription quantitative PCR (RT-qPCR) is considered the ideal method in gene expression studies because of its high sensitivity, time efficiency, and reliability (Derveaux, Vandesompele & Hellemans, 2010; Pfister, Tatabiga & Roser, 2011). In gene expression analysis using RT-qPCR, different starting amounts of messenger RNA between samples and different efficiencies of reverse transcriptases and polymerases can be adjusted by relative quantification, which uses a reference gene (often the housekeeping gene, HKG) as an internal control to calculate target gene (e.g., cytokine gene) expression levels. HKG is required for the maintenance of basic cellular function, and is expressed in all types of cells (Pfaffl, 2004), and its expression level is described as stable. Brinkhof et al. How to cite this article Chen et al (2016), Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas).

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