Quantitative PCR Assay Protocol

SYBR Green reactions were performed using the ABI 7900 HT Fast System (Applied Biosystems, Foster City, CA) in 384 well optical reaction plates. Quantitative PCR (qPCR) assays were performed to measure the expression levels of the target genes as previously described [45 ]. Briefly, aliquots (1 μl) of cDNA (2 ng/reaction) or negative controls were used as template for qPCR reactions with Fast SYBR Green PCR Master Mix (Applied Biosystems) and primers (500 nM final concentration). qPCR amplifications were carried out at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min, and a final dissociation curve analysis step from 65°C to 95°C. Technical replicate experiments were performed for each of the biological samples, in triplicate. Amplification specificity for each reaction was confirmed by the dissociation curve analysis. The Ct values were used for further ΔΔCt analysis. The 16S rDNA was used as a reference gene to normalize samples. A relative quantification value was calculated for each gene with the control group as a reference [27 (link), 40 , 45 ].