Abstract
Groupers are an economically important fish species in world fishery markets. Because many studies using RT-qPCR have addressed gene expression in groupers, appropriate reference genes are required to obtain reliable and accurate results. In this study, the most suitable reference genes were identified from eleven candidate genes of one of the most valuable species, Epinephelus akaara, in a range of different experimental conditions. Using the software packages geNorm, NormFinder, BestKeeper and refFinder, three traditionally used reference genes, β-actin (β-ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-2-microglobulin (B2M), were identified as not suitable for E. akaara gene expression studies, whereas two newly identified reference genes, conserved oligomeric Golgi complex subunit 5 (Cog5) and brefeldin a-inhibited guanine nucleotide-exchange protein 1 (ARFGEF1), could be universally applied under all the tested conditions. These data provide the foundation for more precise results in RT-qPCR studies of gene expression in E. akaara and other Epinephelus species.
Highlights
Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) is a ubiquitous mainstay technology for quantifying gene expression because of its advantages, which include accuracy, specificity, sensitivity, reproducibility and convenience [1, 2]
The overall ranking order of stability of candidate reference genes determined by refFinder was Cog5>navigator 3 (Nav3)>ribosomal protein L17 (RPL17)>Mycbp2>B2M>ARFGEF1>homeodomain-interacting protein kinase 3 (Hipk3)>DHX30>glyceraldehyde-3-phosphate dehydrogenase (GAPDH)>Mgrn1>β-ACT (Fig 2D)
The primary conclusions of this study were as follows: (1) the three traditionally used reference genes, β-ACT, GAPDH and B2M, were not applicable for E. akaara gene expression studies in most situations; (2) the two newly identified reference genes, Cog5 and ARFGEF1, could be universally applied in E. akaara under all tested conditions; and (3) the most suitable reference genes were identified for each specific experimental conditions of E. akaara
Summary
Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) is a ubiquitous mainstay technology for quantifying gene expression because of its advantages, which include accuracy, specificity, sensitivity, reproducibility and convenience [1, 2]. Normalization to a constitutively expressed gene is required to avoid experimental errors caused by different sample amounts, variations in RNA, enzymatic efficiency for cDNA transcription and PCR efficiency [3, 4]. The reference genes that have been used are primarily housekeeping genes, such as β-actin (β-ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tubulin (TUB), elongation factor 1-α (EF1α), polyubiquitin (UBQ) and ribosomal RNAs (18S or 28S rRNA). Selection of new reference genes in Epinephelus akaara collection and analysis, decision to publish, or preparation of the manuscript Many studies show that the transcript levels of these genes vary considerably across cellular conditions [5,6,7,8,9], PLOS ONE | DOI:10.1371/journal.pone.0171646 February 9, 2017
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