Reverse transcriptase quantitative PCR.

Total RNA was extracted using an RNeasy minikit (Qiagen) with on-column DNase digestion using the RNase-free DNase (Qiagen) according to the supplier's instructions. First-strand cDNA was synthesized using a Transcriptor first-strand cDNA synthesis kit (Roche) from 1 μg of total RNA. Using a Bio-Rad CFT Connect real-time system, real-time PCR was performed in 20 μl of a reaction mixture containing FastStart Universal SYBR green master (carboxy-X-rhodamine [ROX]; Roche Applied Science), 0.5 μM each primer, 10 μl of SYBR green PCR master mix, 5 μl of cDNA template, and nuclease-free water. The primers used in this study included HPV16 E7 set 1 (for the H2P, ΔDLYC, CVQ, and ΔLEDLL E7 mutants) primers 5′-AAATGACAGCTCAGAGGAGGAG-3′ (sense) and 5′-GAGTCACACTTGCAACAAAAGG-3′ (antisense) and HPV16 E7 set 2 (for the CKII E7 mutant) primers 5′-TTTGCAACCAGAGACAACTGAT-3′ (sense) and 5′-GAGTCACACTTGCAACAAAAGG-3′ (antisense). β-Actin was detected using the primers 5′-TCACCCACACTGTGCCCATCTA-3′ (sense) and 5′-TGAGGTAGTCAGTCAGGTCCCG-3′ (antisense).