Oligo dt priming

RNA was reverse-transcribed using SuperScript III with oligoDT priming (Invitrogen). Real-time qPCR was performed using iTaq Universal SYBR Green Supermix (BIO-RAD, Hercules, CA) on an ABI 7900HT Real Time PCR System. Primer sequences are listed in Supplemental Table S2.

qRT-PCR analysis of DISC1 transcripts was performed using our standard protocol, and exon specific primers were carefully designed for amplification specificity (Nakata et al., 2009 (link)). Total RNA was extracted from PC12 cells using an RNeasy mini kit (Qiagen). cDNA was synthesized from 1 μg of total RNA using SuperScript III First-Strand Synthesis System with oligo-dT priming (Invitrogen, Grand Island, NY, USA). The qRT-PCR reaction contained 10-fold diluted cDNA from the synthesis reaction, 1 × SYBR GreenER reagent (Invitrogen) and 200 nM specific forward and reverse primers in a 10 μl reaction volume. β-tubulin expression levels were used to normalize the expression data. Primer sequences were as follows: rat DISC1_ex1-forward, 5′-GACAGTGGTTGTCGGCAAGAAT-3′; reverse, 5′-TGCTCCAAGCTACATCAAGGC-3′; rat DISC1_ex10-forward, 5′-TGAGAGAGTGTGGAAAGCGGA-3′; reverse, 5′-AGGCTCTGCATCAGCAA-3′; β-Tubulin-forward, 5′-TGCAGTGGCAAAGTGGAGATT-3′; reverse, 5′-TTGAATTTGCCGTGAGTGGA-3′. Primers were designed using Primer Express 2.0 (Applied Biosystems). The PCR reaction and measurement was carried out with Applied Biosystems PRISM 7900. The PCR reaction conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. A dissociation curve step was added at the end to verify the presence of a single amplicon in the reaction.