qRT-PCR
Total RNA from L3-L5 segments of the spinal cordtissues was extracted with Trizol ( KeyGEN). The cDNA was first synthesized with a reverse transcription reagent kit (Vazyme Biotech, Piscataway, NJ, USA) according to the manufacturer’s instructions. The reagent mixes were prepared with the ChamQ Universal SYBR QPCR Master Mix (Vazyme Biotech), and cDNA was amplified using specific oligonucleotide primers (Table 2). The qRT-PCR was conducted with the PCR System (QTOWER 2.2, Analytik Jena, Germany) using settings of 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and a final 72 °C for 1 min. The relative expression of target genes was calculated using the 2 −ΔΔCT method and normalized to GAPDH.
Table 2. Primer sequences for quantitative real-time polymerase chain reaction in rats.