Total RNA was isolated from TRIzol homogenates of the amygdala in all postmortem subjects. The samples were purified using RNeasy spin columns (Qiagen; Valencia, CA), and RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Walbronn, Germany). cDNA was generated by mixing 1 μg total RNA with oligo-dT primers and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) per the manufacturer’s protocol. PCR products were amplified in quadruplets on a Mastercycler real-time PCR machine (Eppendorf, Hamburg, Germany) using universal PCR conditions, as described previously27. Results were calculated as the geometric mean of threshold cycles of SLC6A4 transcript amplification normalized to three validated internal controls (actin, glyceraldehyde-3-phosphate dehydrogenase, and cyclophilin G). Although performed in serotonergic projection areas, SLC6A4 transcripts are readily detectable by qPCR.
