*2.5. Quantitative Real Time PCR*

Primers were designed with Primer3 tool [45]: forward and reverse primers were always located in two different exons. The primers that were used were summarized in Table S2. The correct amplicon size was verified by 1% agarose-gel electrophoresis. Real-time PCR reactions were performed in 20 μL volume with 1 μL diluted cDNA using the Quantitect® SYBR Green PCR kit (Qiagen) following the manufacturer's instructions. A cDNA pool was serially diluted (from 80 to 2.5 ng per reaction) and used to create standard as well as melting curves and to calculate amplification efficiencies for the primer pair prior to use for quantification. All reactions were performed in triplicates and negative (water) as well as genomic (without reverse transcriptase) controls were always included.
