RT-PCR and quantitative RT-PCR

mRNA was isolated from sympathetic neurons or PC12 cells using TRIzol or RNAeasy mini Kit (QIAGEN) and reverse transcribed with random hexamers and SuperScript III or IV. RT-qPCR reactions (20 μL) contained 10 μL of Flash SybrGreen Mastermix, or 12.5 μL of SybrSelect Mastermix and 0.25 μM primers, unless otherwise indicated. Reactions were performed in duplicate or triplicate with the Mastercycler® Realplex (Eppendorf) or Biorad CFX qPCR machines. For absolute quantification, each experiment included a standard curve, a no-RT control and a no-template control. Standard templates consisted of gel-purified PCR amplicons of known concentrations and each standard curve consisted of seven serial dilutions of the DNA template. For relative quantification, the Comparative Ct Method (ΔΔCt Method) was used. At the end of 40 cycles of amplification, a dissociation curve was performed in which SybrGreen fluorescence was measured at 1°C intervals between the annealing temperature and 100°C. Melting temperatures of amplicons varied between 80°C and 92°C. Primer sequences and PCR conditions are described in Table S4.