Transscript rt reagent kit

Total RNA was extracted from cells using RNAiso Plus (TaKaRa, Japan, 108‐95‐2) and isolated according to the manufacturer's instructions. The RNA concentration and purity were measured by a BioSpectrometer (Eppendorf, Germany); 2 μg total RNA was reversely transcribed into cDNA using the TransScript RT reagent Kit (TransGen, China, AE301). According to the manufacturer's instructions, qRT‐PCR was performed with FastStart Universal SYBR Green Master (Vazyme, China, Q111) using a Gene Amp 9600 PCR system (Perkin‐Elmer, Waltham, MA). The relative amount of cDNA was analyzed using the 2^−ΔΔCT method. The primers for qRT‐PCR used in this study were as follows: PDHA1‐Forward: CTTACCGCTACCATGGACACAGCATG,
Reverse: CTCCTTTAATTCTTCAACACTTGCAAGA; HK2‐Forward: GAGCCACCACTCACCCTACT, Reverse: CCAGGCATTCGGCAATGTG; PFK2‐Forward: ATTGCGGTTTTCGATGCCAC, Reverse: GCCACAACTGTAGGGTCGT; IDH1‐ Forward: TTGGCTGCTTGCATTAAAGGTT, Reverse: GTTTGGCCTGAGCTAGTTTGA; CS‐Forward: GAGCAGGCCAGAGTTAAGAC, Reverse: AAAATAAGCCCTCAGGTAGG; LDHA‐Forward: AAACGCGCCTTAATTTAGTCCA, Reverse:CAGCCGCTTCCAATAATACGG; PGC1α‐Forward: GTAAATCTGCGGGATGATGG, Reverse: AGCAGGGTCAAAATCGTCTG; SIRT1‐Forward: TGCCATCATGAAGCCAGAGA, Reverse: AACATCGCAGTCTCCAAGGA; and GAPDH‐Forward:CAAGAAGGTGGTGAAGCAGG, Reverse: CCACCCTGTTGCTGTAGCC.

Total RNA from virus-infected cells was extracted using an RNA isolation kit (Thermo Scientific, Waltham, MA, USA). First-strand complementary DNA was synthesized from 1 μg of total RNA using a TransScript RT reagent kit (TransGen, Beijing, China), and oligo dT were used for detecting host genes. Generated cDNA was subjected to qPCR in a 25 μL reaction volume using FastStart Universal SYBR Green master mix (Roche, Shanghai, China). Human GAPDH genes were amplified for normalization of the cDNA amount used in qPCR. Reactions were conducted in triplicate, and the data were analyzed using the 2^−ΔΔCt method.