Abstract
In quantitative PCR research, appropriate reference genes are key to determining accurate mRNA expression levels. In order to screen the reference genes suitable for detecting gene expression in tissues of the reproductive axis, a total of 420 (males and females = 1:5) 3-year-old Magang geese were selected and subjected to light treatment. The hypothalamus, pituitary and testicular tissues were subsequently collected at different stages. Ten genes including HPRT1, GAPDH, ACTB, LDHA, SDHA, B2M, TUBB4, TFRC, RPS2 and RPL4 were selected as candidate reference genes. The expression of these genes in goose reproductive axis tissues was detected by real-time fluorescent quantitative PCR. The ΔCT, geNorm, NormFinder and BestKeeper algorithms were applied to sort gene expression according to stability. The results showed that ACTB and TUBB4 were the most suitable reference genes for the hypothalamic tissue of Magang goose in the three breeding stages; HPRT1 and RPL4 for pituitary tissue; and HPRT1 and LDHA for testicular tissue. For all three reproductive axis tissues, ACTB was the most suitable reference gene, whereas the least stable reference gene was GAPDH. Altogether, these results can provide references for tissue expression studies in geese under light treatment.
Highlights
In quantitative PCR research, appropriate reference genes are key to determining accurate mRNA expression levels
Specificity of each of the primers was confirmed by gel electrophoresis and dissociation curve analysis at the expected primer annealing temperature
Quantitative PCR is one of the most powerful techniques used in the detection and determination of nucleic acid content in different types of samples
Summary
In quantitative PCR research, appropriate reference genes are key to determining accurate mRNA expression levels. Ten genes including HPRT1, GAPDH, ACTB, LDHA, SDHA, B2M, TUBB4, TFRC, RPS2 and RPL4 were selected as candidate reference genes. The expression of these genes in goose reproductive axis tissues was detected by real-time fluorescent quantitative PCR. For all three reproductive axis tissues, ACTB was the most suitable reference gene, whereas the least stable reference gene was GAPDH These results can provide references for tissue expression studies in geese under light treatment. An ideal reference gene would show stable expression for a particular tissue type, independent of the developmental stage or experimental treatment conditions Such as ACTB and GAPDH are widely used as common reference genes. The results are expected to provide a reference for gene expression research in reproductive axis tissue of geese under light treatment
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