Quantitative Real-Time PCR of Nuclear Receptor Genes

Total RNA was extracted from neocortical cells that were cultured for 7 DIV (approx. 1.5 × 10⁶ cells per sample) using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The quantity of RNA was spectrophotometrically determined at 260 and 260/280 nm (ND/1000 UV/Vis; Thermo Fisher NanoDrop, USA). Two-step real-time quantitative polymerase chain reaction (qPCR) was performed as previously described [22 (link)]. Both the reverse transcription reaction and qPCR were run on a CFX96 Real-Time System (BioRad, USA). The products of the reverse transcription reaction were amplified using TaqMan Gene Expression Master Mix containing TaqMan primer probes specific to the genes encoding Hprt, Rxrα, Rxrβ, and Rxrγ. Amplification was performed in a total volume of 20 μl containing 10 μl of TaqMan Gene Expression Master Mix and 1.0 μl of reverse transcription product as the PCR template. A standard qPCR procedure was utilized: 2 min at 50 °C and 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The threshold value (Ct) for each sample was set during the exponential phase, and the delta Ct method was used for data analysis. Hprt was used as a reference gene.