Quantitative PCR

Total RNA was extracted from cells using peqGOLD TriFast solution (peqlab Biotechnologie GmbH, Erlangen, Germany) according to manufacturer’s instructions. A total of 2 μg of RNA were reverse transcribed to cDNA in a 20 μl reaction using the High Capacity RNA-to-cDNA Master Mix kit (Applied Biosystems by Life Technologies, Carlsbad, USA), following manufacturer’s instructions. The resulting cDNA was used as template for the quantitative PCR. Quantitative PCR was performed in triplicate, in a total volume of 20 μl, using the primers listed on supplementary table 4 (Table ^S4), and Fast SYBR Green PCR Master Mix (Applied Biosystems by Life Technologies, Carlsbad, USA), according to manufacturer’s instructions. The relative amounts of the PCR products were analysed using the comparative RQ method and using PPIA gene as an internal normalization control.