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.
Highlights
Japanese quail (Coturnix japonica) is the smallest avian species farmed for egg and meat production and is a popular source of proteins in the world [1,2]
To identify RT-qPCR reference genes, we designed primers for ACTB, GAPDH, PGK1, RPL19, RPL32, RPS7, RPS8, SDHA, TBP and YWHAZ based on annotated quail transcripts
R2 values varying from 0.95 to 0.99 (Table 2). qPCR specificity was verified by melting curve analysis (Supplementary Figures S1–S3), agarose gel migration and Sanger sequencing followed by BLAST
Summary
Japanese quail (Coturnix japonica) is the smallest avian species farmed for egg and meat production and is a popular source of proteins in the world [1,2]. In addition to being an established model for embryology studies [3], Japanese quail is a popular laboratory model [4] especially for behavior [5], genetics and genomics studies [6,7]. Since its description in 1992 [10], real-time PCR (qPCR) has been the most common technique to quantify nucleic acid abundance for molecular diagnostics and life science research [11,12,13,14]. Associated with reverse-transcription (RT), this technique became the gold standard to evaluate gene expression
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