Fresh-frozen brain tissue samples dissected from the cortex (Brodmann area 19) were obtained through the Autism Tissue Program (www.atpportal.org) from the Harvard Brain Bank and the NICHD Brain and Tissue Bank at the University of Maryland from 20 samples with a primary diagnosis of autism, and 10 controls. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Complementary DNA (cDNA) was generated from 8μg of total RNA using the Superscript III First-Strand Synthesis kit (Invitrogen). cDNA was diluted 1:5 in 10mM Tris and 1μL of diluted cDNA was used per 10μL PCR reaction. Quantitative real-time PCR was performed on a Lightcycler 480 (Roche Applied Science, Indianapolis, IN) using 2X Taqman Gene Expression Master Mix and probes obtained from Applied Biosystems (ABI, Foster City, CA): SEMA5A (Hs01549381_m1), MAP2 (Hs01103234_g1), TBP (Hs00920497_m1), GAPDH (4333764F). For multiplex reactions, 0.5μL FAM-labeled SEMA5A probe and 0.5μL VIC-labeled MAP2 probe were used per 10μL reaction. The amount of SEMA5A relative to MAP2 was determined for each case using the ΔΔCt method 43. Comparison of SEMA5A to TBP and GAPDH yielded
