Moloney murine leukemia virus reverse transcriptase

Real-time quantitative PCR was carried out with SYBR Green qPCR SuperMix (Bio-Rad) using the CFX-96 system (Bio-Rad). Total cellular RNA was isolated using TRIzol reagent, and cDNA was synthesized from 1 μg of total RNA using oligo-dT and Moloney murine leukemia virus reverse transcriptase (Toyobo, Japan). Relative expression levels of the genes were calculated using the 2^−ΔΔCT method.

The APOT assay was used to amplify HPV oncogene transcripts. The total RNA (1 μg) was reverse transcribed using an oligo (dT)₁₇ RT primer, coupled to a linker sequence (5′-GAC TCGAGTCGACATCGATTTTTTTTTTTTTTTTT’3) (9 (link)). Reverse transcription was conducted for 1 h at 42°C in a reaction system containing 2.5 μM RT primer, 200 units of Moloney murine leukemia virus reverse transcriptase (Toyobo Co., Ltd.), 20 units of RNase Inhibitor (Toyobo Co., Ltd) and 1X RT buffer, in a final volume of 20 μl. In order to open the RNA stem-loop structures, the RNA samples were incubated at 65°C for 10 min prior to mixing with reaction buffer and reverse transcriptase. The cDNA obtained was subsequently amplified by polymerase chain reaction (PCR) in a total volume of 50 μl, using 1 unit of KOD-plus DNA Polymerase (Toyobo Co., Ltd), 0.2 μM HPV-16 E6-specific forward primer P1 (5′-CGACCCAGAAAG TTACCAC-3′) and 0.2 μM P0 reverse primer (5′-GACTCGAGT CGACATCGA-3′). The reaction mixture was subjected to an initial denaturation step for 90 sec, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 59°C for 30 sec, elongation at 68°C for 2 min, and a final elongation step at 68°C for 6 min to ensure the integrity of the amplified fragments.