Construction of Standard Plasmid and Standard Curve of Real-time PCR for Inner Mongolia Cashmere Goats Hairless Gene

Pairs of specific primer were designed according to the conserved coding region of sheep,Bos taurus Hairless and β-actin genome from GenBank.We synthesized the first strand cDNA by reverse transcription PCR after isolated the RNA from the skin tissues.The amplification products were ligated into the pGEM-T vector after recycling and purification,and transformed into competent Top10 cells eventually.Plasmid DNA was purified and identified by restricted enzyme digestion,PCRamplification and sequencing.The positive plasmids enter into the real-time PCR steps after gradient dilution from 103 to 107.The standard curve and regressive curve were generated automatically by the DNA continuous fluorescence detection system.The results showed that no specific amplicons was found according to the derived melting curve of the PCR products, indicating the specification of the present primers.The correlation coefficients of the standard for the Hairless and β-actin genes were 0.998 and 0.999, respectively, suggesting the strong linear relationship between the two groups.The results of repeatability experiments showed that the Ct values hold well for the recombinant plasmids with nine times PCR amplification under the same condition,and the coefficient of variation(CV)was less than 6% among groups(except for 107 copies/reaction),suggesting the great repeatability and stability of the standard curve.Therefore, the construction of the standard plasmid and standard curve established the foundation of the procedures focusing on the quantitation of Cashmere goats Hairless gene mRNA expression in skin tissues.