Quantitative RT-PCR Analysis of Chicken Gene Expression

Total RNA isolated from the 48 chickens was reverse-transcribed using High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) following manufacturer’s instructions. cDNAs was diluted 1:20 and subjected to a Specific Target Amplification step using PreAmp Master Mix kit (Fluidigm Corporation) with a mixture of all primer pairs and 14 cycles of pre-amplification. The BioMark™ 96.96 Dynamic Array (Fluidigm Corporation) for real-time qPCR was used to simultaneously measure the expression of selected genes using Real-Time PCR Analysis User Guide PN 68000088 K1. Primers used for qPCR reactions are listed in Additional file 3. Data were analyzed using HTqPCR R package [15 (link)] and normalized considering GAPDH, RPS8 and TOP2B as reference genes, as suggested after GeNOrm analysis [16 (link)].