Transcription first strand cdna synthesis kit

The cells were collected, and then the total RNA in the cells was extracted using TRIzol reagent (Magen, Guangzhou, China). Total RNA was subjected to reverse transcription (RT) using Transcription First Strand cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China). Real-time quantitative PCR was then carried out in a CFX connect real-time system (Bio-Rad, Hercules, CA, USA) according to the operating instructions of SYBR Green Master Mix (Vazyme Biotech). According to the transcription level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, the gene to be detected was standardized and calibrated. Primer sequences for TRIM25 were used: forward, 5′-TCTGTAGGAGTCAAGGCTAAGGTG-3′, reverse, 5′-GTTGTGGGCGGTATTGTAGTCG-3′. The primer sequences for gRNA, GAPDH, IFNα, and IFNβ have been introduced in previous studies [64 (link)].

To detect the relative mRNA level, a Transcription First Strand cDNA Synthesis kit (Vazyme Biotech) was used to perform the reverse transcription. The reaction conditions were as follows: 50℃ for 15 min and 85℃ for 5 s. A PCR reaction system was prepared using SYBR^®‐Green Real‐Time PCR Master mix (Thermo Fisher Science, ABI7500). The PCR reaction conditions were as follows: 95℃ for 10 s, followed by 40 cycles of 10 s at 95℃ and 30 s at 60℃. GAPDH served as an internal control to normalize the relative expression of GAPDH, PI3K, Akt, GLUT1 and AS160. All data were quantified using the 2‐ΔΔCt method and run‐in triplicate for each sample. The primer sequences used in RT‐qPCR are listed in Table 1.