RNA Isolation and qPCR Analysis

Total RNA isolation was performed as previously described with minor modifications (Vogelaar et al. 2009 (link)). Briefly, after washing the cultures with PBS, explants and adhering axons were collected in lysis buffer, pooling 2–3 wells per sample. After RNA isolation using RNeasy Micro Kit (Qiagen) according to manufacturer’s instruction, samples were subjected to RNase-free DNaseI (Roche) and further purified using the RNeasy MinElute RNA cleanup kit (QIAGEN). cDNA synthesis was performed with the Superscript III First-Strand Synthesis System (Invitrogen) using a mix of random primers and Oligo(dT). Primers (Suppl. Table S1) were designed using the Beacon Designer Software (Premier Biosoft) and optimized on control brain cDNA to determine optimal primer concentration, annealing temperature and efficiency. qPCRs were run in 96-wells plates in the CFX96™ Real-Time PCR Detection System (Biorad). Levels of target genes were calculated in relation to housekeeping genes as previously described (Vogelaar et al. 2009 (link)).