Evaluation of qPCR reference genes in GH-overexpressing transgenic zebrafish (Danio rerio)

Abstract

Reference genes (RGs) must have a stable expression in tissues in all experimental conditions to normalize real-time quantitative reverse transcription PCR (qRT-PCR) data. F0104 is a highly studied lineage of zebrafish developed to overexpress the growth hormone (GH). It is assumed that the transgenic process may influence the expression levels of commonly used RGs. The objective of the present study was to make a comprehensive analysis of stability of canditade RGs actb1, actb2, b2m, eif2s2, eef1a1, gapdh, rplp2, rpl7, rpl13α, tuba1, and rps18, in gh -transgenic and non-transgenic zebrafish. Liver, brain, intestine and muscle samples from both groups had qRT-PCR results analyzed by dCt, geNorm, NormFinder, BestKeeper, and RefFinder softwares. Consensus analyses among software concluded that rpl13α , rpl7 , and eef1a1 are the most stable genes for zebrafish, considering the studied groups and tissues. Gapdh , rps18 , and tuba1 suffered variations in stability among different tissues of both groups, and so, they were listed as the genes with lowest stability. Results from an average pairwise variations test indicated that the use of two RGs would generate reliable results for gene expression analysis in the studied tissues. We conclude that genes that are commonly used in mammals for qRT-PCR assays have low stability in both non-transgenic and gh-transgenic zebrafish reinforcing the importance of using species-specific RGs.

Growth Hormone Overexpression Induces Hyperphagia and Intestinal Morphophysiological Adaptations to Improve Nutrient Uptake in Zebrafish

“…In this study, three candidate reference genes ( elongation factor 1 alpha, eef1α ; β- actin , actb1 ; and acidic ribosomal protein , rpl13a ) were tested using geNorm VBA applet for Microsoft Excel ( Vandesompele et al, 2002 ). The most stable genes were eef1α and rp113a , which were selected as reference genes, in accordance with Rassier et al (2020) . The details on primer sequences and efficiency used in the present study are provided in Table 1 .…”

Diencephalic and Neuropeptidergic Dysfunction in Zebrafish with Autism Risk Mutations

“…Primers for qPCR (Table S1) were designed to span two exons downstream of the target gene mutation and created with all considerations recommended by the manufacturer’s manual. The rpl13a gene was used as the reference with published primers ( 87 ). Raw data from RT-qPCR assays was collected using CFX Maestro Software (Bio-Rad), and analysis of gene expression was performed using the comparative method delta-Ct (dCT)*.…”

New Insights into the Identity of the DFNA58 Gene

“…The mathematical model proposed by PFAFFL [ 45 ], that takes into account the efficiency of the primers, was used in the calculation of the relative quantification. The following reference genes were tested: Actb2 , Ef1 , Rps18 , Sep15 , Metap1 , Rpl13 , and Gapdh [ 46 , 47 ]. Due to the reproducibility of efficiency and relative expression stability among different tissues/organs, the reference genes Actb2 and Rpl13 were selected, and their geometric means or medians were used.…”