Quantitect sybr green based pcr kits

Quiescent cells were infected with HAN or HANΔRNA2.7 ​at an MOI of 1.0 for 72 ​h. DRB was added into the supernatant 4 ​h before cell collection at a final concentration of 200 ​nmol/L. Mock-infected cells were used as controls.
Transcriptions of MCM2, MCM4, MCM5, Cdt1 and Cdc6 were quantified from the cDNA using QuantiTect SYBR Green-based PCR Kits (QIAGEN) on Applied Biosystems 7300 Fast Real-Time PCR System (Applied Biosystem). Primer details are listed in Supplementary Table S2. The reaction conditions were as follows: an initial denaturation at 95 ​°C for 2 ​min, then 40 cycles of annealing/extension at 60 ​°C for 30 ​s followed by a final denaturation at 95 ​°C for 15 ​s. Detections were performed in biological triplicates and relative transcription levels of MCM2, MCM4, MCM5, Cdt1 and Cdc6 were normalized to that of housekeeping gene GAPDH in corresponding samples using 2^−ΔΔCT method.
Lysates were separated by SDS-PAGE. Blotted PVDF membranes were incubated with antibodies against Pol II S2 (Abcam, #ab5095), Cdt1 (Abcam, #ab202067), Cdc6 (Abcam, #ab109315) and GAPDH, followed by peroxidase-conjugated goat anti-mouse or rabbit IgG (ZSGB-BIO, #ZB-2305 or #ZB-2301).

siRNAs transfected HELF cells were infected with HAN or HANΔRNA2.7 ​at an MOI of 1.0. DNA samples were extracted at 2, 24 and 96 hpi, respectively. Quantitative PCR was carried out for HCMV UL83 and GAPDH using QuantiTect SYBR Green-based PCR Kits (QIAGEN, #204243) on Applied Biosystems 7300 Fast Real-Time PCR System (Applied Biosystems). Primer details are listed in Supplementary Table S2. The reaction conditions were as follows: an initial denaturation at 95 ​°C for 2 ​min, then 40 cycles of annealing/extension at 60 ​°C for 30 ​s then denaturation at 95 ​°C for 15 ​s. Detections were performed in biological triplicates and the relative level of viral DNA was normalized to that of housekeeping gene GAPDH in corresponding samples using 2^−ΔΔCTmethods.