Quantifying mRNA Expression with Real-Time PCR

Total RNAs were extracted from cells using an RNA Isolation Kit (Ambion, USA). The reverse transcription reaction was conducted with PrimeScript RT Reagent Kit (TaKaRa, Japan). Quantitative real-time PCR was performed using 2 × ChamQ SYBR qPCR Master Mix (Vazyme, USA). The PCR reaction mixture (10 μL) contained Rox reference Dye, cDNA, ChamQ SYBR qPCR Master Mix (Vazyme), and primers (Table S1). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a reference gene for normalization. The 2^-(△△Ct) method was used to calculate the relative fold change of mRNA expression [20 (link)]. PCR was conducted by maintaining the reaction at 95 °C for 30 s and then alternating for 40 cycles between 95 °C for 5 s and 60 °C for 30 s.