Construction of the recombinant standard plasmids of the GAPDH/β- actin as internal reference genes in Altay sheep using real- time quantitative PCR

To study the real-time quantitative methods for the GAPDH and β-actin in Altay sheep and provide the basis for the sudy of the expression of genes related to functions.A real-time fluorescence quantitative PCR method was used to clone the partial sequences of sheep GAPDH and β-actin genes,and the conventional PCR product was used as the standard to establish a standard curve and melting curve.The results showed that the correlation coefficients of the standard curve were 0.999 9 and 1.000 0,respectively; and the amplification efficiencies were 96% and 109%,respectively.There were sharp peaks in the melting curve,and there was no non-specific miscellaneous peak,indicating a single PCR product,no primer dimers and non-specific amplification products.The results indicate that the established methd has strong specificity with high correlation coefficient,and can be applied to study of the quantitative analysis for the functional gene expression in Altay sheep.