Cfx96 touch rt pcr machine

Total RNA was extracted from HEK293 or PAE cells and DNase-treated using the RNeasy Mini kit (Qiagen). Total RNA was extracted for mouse brains and DNase-treated using the RNeasy Lipid Tissue Mini kit (Qiagen). cDNA was synthetized using the iScript™ cDNA Synthesis Kit (Bio-Rad). Real-time quantitative PCR was performed on a CFX96 Touch RT-PCR machine (Bio-Rad) using either SYBRgreen (iTaq Universal SYBR Green Supermix, Bio-Rad) or TaqMan probes (Life Technologies). In all conditions, eukaryotic 18S rRNA (4319413E) was used as a reference gene for ΔΔCt calculations. Since HEK293 cells express detectable levels of human PDGFB mRNA, their expression level was taken as 1 and the other values are expressed as fold increase over basal expression. When using SYBRgreen, the following primers were used. For detection of human PDGFB: forward primer 5’-GAT GAT CTC CAA CGC CTG-3’ and reverse primer 5’-TCC TTC TTC CAC GAG CCA-3’. For detection of human PDGFRB: forward primer 5’-ACC TGC AAT GTG ACG GAG GAG-3’ and reverse primer 5’-AAC ACT ACC TGC AGT GTC CG-3’. The following TaqMan probes were used: Mouse Pdgfrb (Mm00435546_m1) and mouse Pdgfb (Mm00440677_m1). All RT-PCR data is represented as the mean of three independent experiments.
The Quantikine ELISA Kit (R&D Systems) was used to measure the concentration of PDGF-BB in the conditioned media.