qPCR Analysis of Autophagy-Related Genes, Nuclear Receptors, and miRNAs

Total RNA was purified from 7 DIV neocortical cells using the RNeasy Mini Kit or the miRNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions as previously described [25, 28–30]. The RNA quantification was spectrophotometrically determined at 260 nm and 260/280 nm (ND/1000 UV/Vis; Thermo Fisher NanoDrop, USA). cDNA was synthesized using the High Capacity cDNA–Reverse Transcription Kit (Thermo Fisher Scientific, USA) or the miScript II RT Kit (Qiagen, Valencia, CA). Both the reverse transcription reaction and qPCR were performed on a CFX96 Real-Time System (Bio-Rad, Hercules, CA, 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, Becn1, Nup62, Atg7, Map1lc3a, Map1lc3b, Rxrα, Rxrβ, Rxrγ, Pparγ, SNORD95, miR-19b, miR-33, miR-489, and miR-509. 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 (C_t) for each sample was set during the exponential phase, and the delta C_t method was used for data analysis. To evaluate the reference gene expression, RefFinder web-based comprehensive tool has been used. Hprt (the gene encoding hypoxanthine phosphoribosyltransferase) was selected to use as a reference gene against Becn1, Nup62, Atg7, Map1lc3a, Map1lc3b, Rxrα, Rxrβ, Rxrγ, and Pparγ. SNORD95 (the small nucleolar RNA, C/D box 95) has been chosen to be a reference gene in the cases of miR-19b, miR-33, miR-489, and miR-509. The results were obtained from three independent experiments.