Gene expression assays for NTR and NESDA were conducted at the Rutgers University Cell and DNA Repository. Total RNA was extracted at Rutgers (for NESDA, at VU Medical Center) using the PAXgene Blood RNA MDx Kit protocol in 96 well format using the BioRobot Universal System (Qiagen). RNA quality and quantity was assessed by Caliper AMS90 with HT DNA5K/RNA LabChips. Samples were randomized to plates, with checks to ensure sex/zygosity balance. Co-twins were randomized without respect to relationship to avoid bias in family correlation estimates. For cDNA synthesis, 50ng of RNA was reverse-transcribed and amplified in a plate format on a Biomek FX liquid handling robot (Beckman Coulter) using Ovation Pico WTA reagents (NuGEN). Products purified from single primer isothermal amplification (SPIA) were fragmented, labeled with biotin (Encore Biotin Module, NuGEN), and size distributions verified (Caliper AMS90, HT DNA 5K/RNA LabChips). Samples were hybridized to Affymetrix U219 (Supplementary Note) array plates to enable expression profiling in 96-sample sets. Array hybridization, washing, staining, and scanning were carried out in an Affymetrix GeneTitan System per the manufacturer’s protocol.
