S5 DNA Methylation testing
Methylation assays were based on end-point PCR and quantitative pyrosequencing of amplicons using primers for late regions L1 and L2of HPV16, HPV18, HPV31 and HPV33 combined with the promoter region of human tumor suppressor gene EPB41L3. Briefly, genomic DNA was extracted using 100 μL of suspensions with a QIAamp DNA Micro Kit (Qiagen, Hilden, Germany) and DNA was quantified by UV absorption. 250 ng of DNA was used in the bisulfite treatment where un-methylated cytosines were converted to uracil with the EpiTect Fast Bisulfite Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Converted DNA was eluted in 15 μL Buffer EB twice and the eluant was combined for next steps. Converted DNA (2μL/sample) was added to the PCR mix system with 12.5 μL of 2× PyroMark PCR master mix (Qiagen) and optimized concentrations of primers and Magnesium chloride. Polymerase chain reaction was conducted on a Bioer Technology LifeECO amplication instrument (TC-96/G/H(b)C model, Hangzhou Bioer Technology Co., Ltd.). The PCR products were used for the followed pyrosequencing (PyroMarkQ48 Autoprep, Qigen, Hilden, Germany). The methodological details were detailed in several studies13. The laboratory was blinded to HPV genotyping results before the specimen was performed S5 methylation assay to minimize these unintentional biases.