Real-Time Quantitative PCR

The primers based on the chicken POMC gene sequences (GenBank NO.{"type":"entrez-nucleotide","attrs":{"text":"NC_006090.5","term_id":"1375922356","term_text":"NC_006090.5"}}NC_006090.5) for qPCR were shown in Table 2, and chicken β-actin gene was used as the internal control. The qPCR assays were performed using the Takara SYBR Premix Ex Taq II kits (Takara Bio,China) on a 96-well Applied Biosystems 7,900 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). The 10 μL reaction mix included 1.0 μL of cDNA template, 5 μL of SYBR PremixExTaq II, 0.4 μL of forward and reverse primers, 0.2 μL of Rox Reference Dye II, and 3 μL of sterile water. Detection of each sample was performed simultaneously 3 times. The procedure for qPCR was as follows: predenaturation at 95°C for 30 s，followed by 40 cycles of 95°C for 5 s, and 60°C for 30 s. The mRNA expression levels were normalized to the chicken β-actin gene and calculated using the 2^−ΔΔCt method.

Primers used for real-time quantitative PCR of POMC gene.