Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus).

I-Hua Chen,Myra Blanchard,Jeffrey Stott,Shih-Jen Chou,I-Fan Jen,Wei-Cheng Yang,Lien-Siang Chou,Jiann-Hsiung Wang

doi:10.1038/srep15425

Abstract

Quantitative RT-PCR is often used as a research tool directed at gene transcription. Selection of optimal housekeeping genes (HKGs) as reference genes is critical to establishing sensitive and reproducible qRT-PCR-based assays. The current study was designed to identify the appropriate reference genes in blood leukocytes of bottlenose dolphins (Tursiops truncatus) for gene transcription research. Seventy-five blood samples collected from 7 bottlenose dolphins were used to analyze 15 candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ, LDHA, SDHA). HKG stability in qRT-PCR was determined using geNorm, NormFinder, BestKeeper and comparative delta Ct algorithms. Utilization of RefFinder, which combined all 4 algorithms, suggested that PGK1, HPRT1 and RPL4 were the most stable HKGs in bottlenose dolphin blood. Gene transcription perturbations in blood can serve as an indication of health status in cetaceans as it occurs prior to alterations in hematology and chemistry. This study identified HKGs that could be used in gene transcript studies, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans.

Highlights

Quantitative reverse transcription PCR represents a rapid and reliable method for the detection and quantification of mRNA transcripts of a selected gene of interest (GOI) and is well suited to study biological processes, many of which can have practical clinical applications[1]
These studies have been based upon Quantitative reverse transcription PCR (qRT-PCR) using a variety of different Housekeeping genes (HKGs), including ribosomal protein L8 (RPL8) in killer whale (Orcinus orca) skin biopsies[9], glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tyrosin 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) in harbor porpoise (Phocoena phocoena) blood samples[10,11,12], GAPDH in bottlenose dolphin (Tursiops truncatus) blood samples[13], and RPS9 in blood samples in bottlenose dolphins, beluga whales (Delphinapterus leucas), and Pacific white-sided dolphins (Lagenorhynchus obliquidens)[8,14]
The same 10 HKGs were tested in transcript analysis of striped dolphin fibroblast cultures exposed to organochlorines (OCs), polybrominated diphenyl ethers (PBDEs) and 17β -estradiol[16]

Summary

Introduction

Quantitative reverse transcription PCR (qRT-PCR) represents a rapid and reliable method for the detection and quantification of mRNA transcripts of a selected gene of interest (GOI) and is well suited to study biological processes, many of which can have practical clinical applications[1]. Cetacean gene transcripts have been the subject of quantitative analysis in multiple tissues and species These studies have been based upon qRT-PCR using a variety of different HKGs, including ribosomal protein L8 (RPL8) in killer whale (Orcinus orca) skin biopsies[9], glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tyrosin 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) in harbor porpoise (Phocoena phocoena) blood samples[10,11,12], GAPDH in bottlenose dolphin (Tursiops truncatus) blood samples[13], and RPS9 in blood samples in bottlenose dolphins, beluga whales (Delphinapterus leucas), and Pacific white-sided dolphins (Lagenorhynchus obliquidens)[8,14]. Acquisition of such data is central to our long-term goal of applying qRT-PCR in multidisciplinary immune assessments of captive and free-ranging cetacean species

Methods

Results

Conclusion