Quantitative RT-PCR Analysis of aSyn

Total mRNA was isolated with RNeasy Mini Kit (Qiagen, Venlo, Netherlands, #74104), following the manufacturer's instructions. One microgram was used to synthetize cDNA (20 μl reaction volume) using the Omniscript reverse transcriptase kit and oligo-dt primers (Qiagen, # 205111 and #79237) in a Thermo Hybaid PCR Express (Fischer Scientific, Schwerte, Germany). Real-time PCR analyses of 1 μl of the cDNA products were performed in duplicate with iQ SYBR Green Supermix (Bio Rad, Munich, Germany, #170-8882) in a CFX96 Real time system Thermal cycler (Bio Rad). The primer pairs used for real-time amplification of aSyn are described in Rhinn et al.^{59 (link)} Primers for HPRT^{41 (link)} and QuantiTect Primer assay GAPDH mix (Qiagen #QT00079247) were used for normalization. Relative expression levels were calculated with subsequent ΔCT values that were assessed using the comparative CT method with subsequent ΔCT values normalized to the housekeeping genes HPRT and GAPDH.