Quantitative RT-PCR Analysis of Gene Expression

Total RNA was isolated from the cells using a commercially available kit (NucleoSpin RNAII from Macherey–Nagel, Dueren, Germany), as previously described^{25 (link)}. The quantity and purity of the RNA were measured with a Nanodrop ND-1000 spectrophotometer (Nyxor Biotech, Paris, France). Total RNA (1 μg) was reverse-transcribed into first-strand cDNA using a High-Capacity cDNA Achieve Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. RT-qPCR was performed using the fluorescent dye SYBR Green method, with SYBR Green PCR Master Mix in 384-well plates and the StepOnePlus system (Applied Biosystems). Amplification curves were analyzed according to the comparative cycle threshold method, using StepOnePlus software (version 2.1, Applied Biosystems by Life Technologies, Paisley, UK). The steady-state mRNA levels for the genes of interest were normalized against those of GAPDH.