Pyromark q96

Nine pairs of cervical cancer specimens and adjacent normal cervical tissues were obtained from the Second Affiliated Hospital of Wenzhou Medical University. Cervical cancer patients were diagnosed by experienced pathologists based on the results of surgically removed specimens (Supplementary Table 1). Human genomic DNA was extracted from tissue samples using the Genomic DNA Extraction Kit (Qiagen, Dusseldorf, Germany). DNA concentrations were determined by the Infinite F200 Tecan microplate reader (Tecan, männedorf, Switzerland). Primers were designed using the PyroMark Assay Design Software 2.0 and bisulfite-treated DNA PCR-amplified using the PyroMark PCR kit prior to analysis on a PyroMark Q96 according to manufacturer’s instruction (Qiagen, Dusseldorf, Germany). Sequences of the PCR primers were shown in Table 3. Amplification was carried out as follows: 95°C for 3 min, followed by 40 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min, with a final elongation step at 72°C for 7 min. Raw data were analyzed using PyroMark Q96 software (Qiagen, Dusseldorf, Germany). The research protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University. Written informed consents were obtained from all subjects.

Blood DNAs from 17 Rb patients (including 2 carriers of the p.Arg661Trp mutation and 6 carriers of a large deletion of known parental origin) and 2 controls underwent bisulfite conversion using the EZ DNA Methylation-Gold kit (Zymo research) according to the manufacturer’s instructions. Pyrosequencing primers were designed to cover the whole CpG85 island using the PyroMark Assay Design Software v1.0.6 (Qiagen). Bisulfite conversion was assessed by PCR amplification using converted DNA specific primers and agarose gel electrophoresis. Samples were prepared with the PyroMark Q96 Vacuum Workstation and pyrosequencing was performed on a PyroMark Q96 (Qiagen) according to the manufacturer’s instructions with subsequent analysis using the Analysis software package v2.5.7 (Qiagen).

Quantitative pyrosequencing was performed with the PyroMark PCR Kit (Qiagen). Predesigned methylation assays were used to determine the methylation status of three CpG sites in the RASSF1A and six CpG islands in the tissue inhibitor of metalloproteinase‐3 (TIMP3) promoter (PyroMark CpG assay; Qiagen). The total volume of PCR reaction was 25 μl including 20 ng of bisulphite‐treated DNA. Thermal cycling protocol included steps as initial denaturation at 95°C for 15 min., followed by 45 cycles of amplification: 94°C for 30 sec., 56°C during 30 sec. and 72°C for 30 sec. and a final extension at 72°C for 10 min. The amplification products were confirmed by electrophoresis on 1.75% agarose gel, stained with GelRed Nucleic Acid Biotium Inc. (Fremont, CA, USA) and visualized on UV transilluminator. Obtained PCR products were analysed according to the manufacturer's instructions using the PyroMark Q96 ID System (Qiagen) with PyroMark Gold Q96 Reagents. Methylation data were evaluated with the instrument software (PyroMark Q96 software, version 2.5.8; Qiagen).