Quantitative RT-PCR Analysis of Gene Expression

RNA was isolated by using PureLink RNA Mini Kit (Thermo-Fisher) following manufacturer’s recommendations. One to two micrograms of RNA were reverse transcribed in a 20 μl reaction using oligo(dT)^{20 (link)} and SuperScript III First-Strand Synthesis System (Thermo-Fisher). Relative quantitative real-time PCR was performed in a total reaction volume of 10 μl, using 2 μl (20 μg/μl) cDNA, 1 μl gene specific primer mix (QuantiTect primer Assays), 5 μl SYBR GreenER qPCR SuperMix (Thermo-Fisher), and 2 μl water. The quantification of gene expression was performed using 7900HT Fast Real-Time PCR System (Applied Biosystems) in triplicate. The thermal profile of the reaction was: 50 °C for 2 min, 95 °C for 10 min and 45 cycles of 95 °C for 15 s followed by 60 °C for 1 min. Amplification of the sequence of interest was normalized with a reference endogenous gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The value was expressed as a fold change relative to RNA from cells infected with control adenovirus (Ad. Null), control siRNA (siControl), or non-treated cells. For data analysis, the 7900HT Fast Real-Time PCR System Software was used (Applied Biosystems).