SHORT-COMMUNICATION Validation of reference genes for real-time quantitative PCR in tambaqui (Colossoma macropomum)

Abstract

Tambaqui, Colossoma macropomum, is the main native freshwater fish in Brazilian aquaculture. Therefore, intensive research pressure has been applied to the species to support new technologies for tambaqui farming. Molecular biology represents a tool that can be used to investigate every field of applied biology, from fish physiology to the effects of climate change. Based on the importance of reference genes for the relative or absolute quantification of gene transcripts, we cloned and sequenced three candidate reference genes in tambaqui (18S ribossomal RNA - 18s, glyceraldehyde-3-phosphate dehydrogenase - gapdh, and actin beta - β-actin), and validated a set of primers for each gene for use in real-time quantitative PCR. The results were evaluated by RefFinder, which indicated that β-actin is the most suitable reference gene for tambaqui among those studied, followed by 18s.

Regulation of gene expression associated with LC‐PUFA metabolism in juvenile tambaqui ( Colossoma macropomum ) fed different dietary oil sources

“…The sequences of the key gene involved in the LC‐PUFA biosynthesis ( fads2 , elovl5 and elovl2 ) and TF ( pparαa , pparαb , pparβa , pparβb , ppary , lxrα and srebp1 ), and enzymes involved in the fatty acid biosynthesis ( fas ) and lipid catabolism ( lpl ), were retrieved from tambaqui liver and brain transcriptome assemblies (Machado et al, 2019). For the normalization of the real‐time quantitative PCR, we combined β‐actin ( actb ), a stable housekeeping gene in tambaqui (Nascimento et al, 2016), with α‐tubulin ( tuba ) and β‐2‐microglobulin ( b2 m ).…”

Phylogenomic and expression analysis of Colossoma macropomum cyp19a1a and cyp19a1b and their non-classical role in tambaqui sex differentiation

“…The qPCR reactions were performed in the 7500 Fast Real-Time PCR System v2.3 (Applied Biosystems), in duplicates, using 2 µL of cDNA (120 ng), 1 µL of each primer (200 nM), 12,5 µL Fast SYBR Green PCR Master Mix (Applied Biosystems), and nuclease-free water to a final volume of 25 µL. qPCR data was analyzed using the 2 -(ΔΔCt) method where the expression of the target gene was first normalized to the β-actin (Nascimento et al, 2016) and then calibrated to the mean of the pool of larvae (first sampling) for the non-treated fish (i.e. differentiating juveniles and gonads of adult tambaqui) and of the control group in the treated fish (experimental groups).…”

Diurnal and seasonal hsp70 gene expression in a cryptic reef fish, the bluebanded goby Lythrypnus dalli (Gilbert 1890)

“…Specific forward and reverse primers for both hsp70 and the reference gene β-actin were designed (~150 bp amplicon length, ~55 % GC content, 60 °C primer T m ) using Primer3v.0.4.0 (Untergasser et al, 2012). β-actin is one of the most commonly used reference gene to quantify mRNA and its stability has been analyzed in some fish species under thermal stress conditions (Ma et al, 2019;Nascimento et al, 2016;Purohit et al, 2015). The primers were F: 5-GAGAAC-GTGCAGGACCTGCT-3, R: 5-CCGGGCTGGTTGTCGAAGTA-3 for hsp70; and F: 5-TGAAGATCCTGACAGAGCGTGGC-3, R: 5-TTGCCGATGGTGATGACCTGT -3 for β-actin.…”