Real-Time qPCR Standardization Protocol

For each transcript a standard curve was constructed using the purified PCR product generated for each specific primer pair. Single reactions were prepared for each cDNA along with each serial of dilution using the Brilliant^® SYBR^® Green Master Mix (Stratagene). Each PCR reaction also included a reverse transcription negative control (without reverse transcriptase) to confirm the absence of genomic DNA, a non template negative control to check for primer-dimer and a porcine genomic DNA control to verify no specific amplification with the primers. Each reaction consisted of 20 μl containing 2 μl of cDNA and 5 pmol of each primer. The real time qPCR was run on MX3000p (Stratagene). The cycling conditions were 1 cycle of denaturation at 95°C/10 min, followed by 40 three-segment cycles of amplification (95°C/30 sec, 58°C–63°C (gene depending, see table 2)/1 min, 72°C/30 sec) where the fluorescence was automatically measured during PCR and one three-segment cycle of product melting (95°C/1 min, 55°C/30 sec, 95°C/30 sec). The baseline adjustment method of the Mx3000 (Stratagene) software was used to determine the Ct in each reaction. A melting curve was constructed for each primer pair to verify the presence of one gene-specific peak and the absence of primer dimmer. All samples were amplified in duplicates and the mean was used for further analysis.