cDNA was reverse transcribed using the Ready-To-Go Kit from Amersham Bioscience (Piscataway, NJ) following manufacturer’s instructions. ZNF804A expression was analyzed on the StepOne Plus instrument (ABI, Foster City, CA) using a Taqman quantitative real-time PCR expression assay (ABI, Foster City, CA). Each sample was assayed in triplicate and mean values from the triplicates were used for all analyses. Gene expression was normalized against two reference genes47 (HPRT and TBP), and analyzed by the 2−ΔΔCT algorithm48. The 2−ΔΔCT algorithm, widely used to compare differences in gene expression, is valid only if PCR efficiency of studied genes is equal48. PCR efficiencies (measured by standard curve) of all three genes assayed were very similar (ZNF804A: 91%, HPRT: 93%, TBP: 90%).
