Identification of Reference Genes for Quantitative Gene Expression Studies in Three Tissues of Japanese Quail

Abstract

RT-qPCR is the gold standard for candidate gene expression analysis. However, the interpretation of RT-qPCR results depends on the proper use of internal controls, i.e., reference genes. Japanese quail is an agronomic species also used as a laboratory model, but little is known about RT-qPCR reference genes for this species. Thus, we investigated 10 putative reference genes (ACTB, GAPDH, PGK1, RPS7, RPS8, RPL19, RPL32, SDHA, TBP and YWHAZ) in three different female and male quail tissues (liver, brain and pectoral muscle). Gene expression stability was evaluated with three different algorithms: geNorm, NormFinder and BestKeeper. For each tissue, a suitable set of reference genes was defined and validated by a differential analysis of gene expression between females and males (CCNH in brain and RPL19 in pectoral muscle). Collectively, our study led to the identification of suitable reference genes in liver, brain and pectoral muscle for Japanese quail, along with recommendations for the identification of reference gene sets for this species.

Embryonic methionine triggers post-natal developmental programming in Japanese quail

“…We normalised relative changes in gene expression against RPL19 gene expression as the most stabile reference gene selected from 6 housekeeping genes such as ACTB , GAPDH , RPL19 , RPS8, 18S , and RPL13 by 3 algorithms (delta Ct, Best Keeper, NormFinder) (Simon et al 2018 ; Vitorino Carvalho et al 2019 ). Relative gene expression compares the expression level of gene of interest or the target gene to the expression level of the reference gene housekeeping genes that are usually expressed relatively constant in all cells and most of the conditions (Joshi et al 2022 ).…”

Can increased prenatal exposure to thyroid hormones alter physiology and behaviour in the long-term? Insights from an experimental study in Japanese quails

“…CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. GAPDH and PGK were used as control genes, using primers previously designed and validated in Japanese quail (Vitorino Carvalho et al, 2019). Stability of expression between experimental groups was further verified using geNorm software (geNorm M < 0.5, geNorm V < 0.15), and thus the geometric mean of these two genes was used as our reference gene.…”

Can Increased Prenatal Exposure to Thyroid Hormones Alter Physiology and Behaviour in the Long-Term? Insights from an Experimental Study in Japanese Quails

“…GAPDH and PGK were used as control genes, using primers previously designed and validated in Japanese quail (Vitorino Carvalho et al, 2019). Stability of expression between experimental groups was further verified using geNorm software (geNorm M < 0.5, geNorm V < 0.15), and thus the geometric mean of these two genes was used as our reference gene.…”