Quantitative PCR Protocol for Gene Expression

Transcript levels of genes were determined by quantitative PCR with the SYBR Green method on a Rotor-Gene Q (Qiagen). A 15 μl reaction mixture contained the following components: 5 μl cDNA, 1 μl of each primer (5 μM stock), 1.5 μl buffer, 0.3 μl dNTPs (10 mM each), 5.75 μl water, 0.3 μl JumpStart Taq DNA polymerase and 0.15 μl CYBR Green (1:400 dilution of purchased stock solution of ABsolute™ QPCR SYBR Green Fluorescein Mix, ABgene). The thermal cycling conditions used were 95 °C for 6 min followed by 40 cycles of 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 20 s, followed by a melt cycle with 1 °C increments for 5 s each from 56 to 96 °C. The analysis of melting curves, measurement of primer pair efficiencies, and determination of cycle threshold values, including the calculation of the mean normalized expression of the genes, was conducted using the Rotor-Gene Q Series Software Q 2.0.2 (Qiagen) and the Q-Gene software. The mRNA levels of the studied genes were normalized using the comparative C_T method, using the expression of one or two reference genes (actin and ribosomal protein L19, RPL19) as internal standard (Maroufi et al., 2010 (link); van Arkel et al., 2012 (link)). Primer efficiency was considered valid when calculated efficiency was between 90 and 110% with 100% as an optimum.