pgRNA in cell-culture supernatant was detected by RT-PCR. Total RNA from the supernatants was extracted by using RNA extraction kit (Takara, China) and reverse transcribed using a PrimeScript™ RT reagent Kit to produce cDNA; RT-PCR was performed to detect pgRNA using Premix Ex Taq™ (Takara, China) using the primers 5’-CACCTCTGCCTAATCATC-3’ and 5’-GGAAAGAAGTCAGAAGGCAA-3’.
The cccDNA levels in cells were quantified by quantitative polymerase chain reaction (qPCR). Total DNA was extracted from the cells using a TIANamp Genomic DNA Kit (Tiangen, China) following DNase reagent (Takara, China) to enhance the efficiency of the specific extraction of cccDNA. qPCR was performed using primers 5’-GACTCCCCGTCTGTGCCTTCTCATC’, and 5’-AGACCAATTTATGCCTACAGCCTCC-3’ for cccDNA amplification using SYBR Premix Ex Taq (Takara, China).
Amplification was performed as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 34 s, 95 °C for 15 s and 60 °C for 1 min.
The cccDNA levels in cells were quantified by quantitative polymerase chain reaction (qPCR). Total DNA was extracted from the cells using a TIANamp Genomic DNA Kit (Tiangen, China) following DNase reagent (Takara, China) to enhance the efficiency of the specific extraction of cccDNA. qPCR was performed using primers 5’-GACTCCCCGTCTGTGCCTTCTCATC’, and 5’-AGACCAATTTATGCCTACAGCCTCC-3’ for cccDNA amplification using SYBR Premix Ex Taq (Takara, China).
Amplification was performed as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 34 s, 95 °C for 15 s and 60 °C for 1 min.