Real-Time Quantitative RT-PCR Assays (RT-qPCR)

Real-time RT-qPCR was used to validate the microarray data. Amplification was carried out using a 7500 Fast System (Applied Biosystems). RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems). The specific primers used for the RT-qPCR assays were designed with the Primer Express 3.0 software and listed in the Supplementary Table S1. The SYBR Green method was used and each assay was performed in triplicate using SYBR Green real-time PCR Master Mix (Applied Biosystems). Amplification was initiated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Control PCRs were included to confirm the absence of primer dimer formation (no-template control), and to verify that there was no DNA contamination (without RT enzyme negative control). All real-time PCR assays amplified a single product as determined by melting curve analysis and by electrophoresis. A standard curve was plotted with cycle threshold (Ct) values obtained from amplification of known quantities of cDNAs and used to determine the efficiency (E) as E = 10^–1/slope. The expression levels of target genes were normalized. The Bestkeeper analysis (Pfaffl et al., 2004) was applied, and the geometric mean of the most stably expressed housekeeping genes (16S rRNA, gapB, dnaG, and gyrA) was used as a normalization factor. The expression ratios measured by microarrays and by RT-qPCR assay were plotted, and the linear correlation coefficient was calculated (y = 0.9507x + 0.1473; R² = 0.9758).