Quantstudio 3 real time pcr system

Messenger Ribose Nucleic Acid (mRNA) levels were examined by routine real‐time qPCR assay. Total Ribose Nucleic Acid (RNA) from cells and tissues were extracted, respectively, using TRIzol kit (Thermo Fisher). One‐microgram RNA was used for reverse transcription using the PrimeScript RT reagent with gDNA Eraser (Takara; cat# RR047A). qPCR was performed with iQ SYBR Green Supermix (Bio‐Rad; cat# 1708882) and QuantStudio 3 Real‐Time PCR System (Bio‐Rad). Relative mRNA expression was normalized with Actb. Primer sequences are shown in Table S2.

Total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. After cDNA quantification by NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA), qRT-PCR was performed using Universal iTaq SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer’s instructions and using a QuantStudio™ 3 Real-Time PCR System. We used 5 µL SYBR Green Mix (2×), 3 µL of 600 nM forward and reverse primers mix (Thermo Fisher Scientific, Waltham, MA, USA) and 2 µL of 90–100 ng/mL cDNA sample per reaction. Cycles consisted of polymerase activation and DNA denaturation at 95 °C for 3 min, 40 cycles of amplification and annealing–extension at 95 °C for 15 s followed by 60 °C for 1 min, respectively. All primers used for gene expression analysis are described in Table 1. GAPDH was used as a housekeeping gene to calculate the relative quantification by the ∆∆C_T method.