RNA Extraction and qPCR Analysis in Hypoxia/Ischemia

Total RNA (approximately 1 µg of RNA per sample) was isolated from brain tissues 3 days after hypoxia/ischemia using the RNeasy Mini Kit (Qiagen, Valencia, CA). The quantity of RNA was spectrophotometrically determined at 260 nm and 260/280 nm (ND/1000 UV/Vis; Tecan NanoDrop, USA). cDNA was synthesized using the High Capacity cDNA-Reverse Transcription Kit (Thermo Fisher Scientific, USA). The reverse transcription reaction and quantitative polymerase chain reaction (qPCR) were run on the CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA) as previously described [21 (link)]. 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. To evaluate reference gene expression, the RefFinder web-based comprehensive tool was used [22 (link)]. For our study, NormFinder, BestKeeper and delta Ct recommended Hprt as the most stable reference gene.