Aceq qpcr sybr green master mix sybr green 1 kit

rAAV2 was transfected into HeLa cells at 5000 vgs/cell for 12 h, followed by isolation of cytoplasmic and nuclear fractions. In line with specific protocols, total DNA of cytoplasmic and nuclear fractions were collected with Qiagen DNeasy blood and tissue kit. Human GAPDH gene was the nuclear reference genes. Cell internal reference genes and viral genomes were analysed based on the previous description.³³ We used all primers (Table S1) for quantifying GFP, EGFR and GAPDH. All the reactions were run with AceQ qPCR SYBR Green Master Mix (SYBR Green I) kit (Vazyme Biotechnology) on ABI Step One Fast Real‐Time PCR System. PCR conditions were shown as following: 10 min at 95°C; 10 s at 95°C, 10 s at 60°C and 10 s at 72°C for 45 cycles. For absolute quantification, second‐derivative maximum comparisons were made against the plasmid DNA standard curves.

The HeLa cells were transduced with ss‐rAAV2‐GFP or ss‐rAAV2‐GFP‐miRNA133b in 6‐well plates for 48 h. We utilized Trizol for total RNA extraction, which was later prepared into cDNA by Primer Script RT reagent Kit (TaKaRa Bio) via reverse transcription. All the experimental procedures were performed at low temperature. The miRNA expression was examined using AceQ qPCR SYBR Green Master Mix (SYBR Green I) kit (Vazyme Biotechnology) with an ABI Step One Fast Real‐Time PCR System by applying gene‐specific primers (Table S1) in line with specific protocols. Relative content of miRNA was normalized to endogenous U6 small nuclear RNA.