Pyrogold q96 sqa reagents

Templates for PSQ were prepared by amplifying bisulfite modified DNA with a forward primer (GTTTYGGATATGTTGGGATAG) and a biotinylated reverse primer (AAAACCACTCRAAACTACCAC). Two assays were designed and run on this template using two PSQ primers: GATAGTTYGYGTTTTTAGAA (assay for CpGs 74–83) and GYGATTTGGTGAGTGTTTG (assay for CpGs 84–89). PSQ was performed using PyroGold Q96 SQA Reagents and the Pyro Q-CpG software on a PyroMark ID pyrosequencer (Qiagen, Crawley, UK) as per manufacturer’s recommendation. Full details for CpG location and PSQ can be found in Malley et al. [6 (link)] and Mullolland et al. [11 (link)].

Bisulfite conversion and pyrosequencing was carried out according to Sun et al (34 (link)) with the following modifications: gDNA (1 μg) was treated using the Imprint DNA modification kit (Sigma) in accordance with the manufacturer's instructions for the two-step conversion and eluted in 20 μL. Primer sequences, annealing temperatures, and cycle number are shown in Supplemental Methods. Pyrosequencing was performed on the PSQ HS96 system using PyroGold Q96 SQA reagents (QIAGEN). The degree of methylation at CpG sites (without distinguishing between maternal and paternal alleles) was determined by pyro-Q CpG software (Biotage). Information about the sequence and CpG groups analyzed can be found in Supplemental Methods.

Promoter methylation status of the MGMT gene was conducted with a PCR-based method using DNA treated with bisulfite followed by a real-time pyrosequencing that targeted CpG islands (EpigenDxInc). This method allowed us to quantify methylation at multiple CpG sites individually. Pyrosequencing was performed using PyroGold Q96 SQA Reagents and the Pyro Q-CpG software on a PyroMark ID pyrosequencer (Qiagen) as per the manufacturer’s recommendation. The sequencing results were analyzed using the PSQ PyroMark software (Qiagen). As controls, CpGenome Universal Methylated DNA (positive methylation control; Chemicon International) and DNA from FFPE non-tumorous skin tissue (negative methylation control) were included in the assay, as well as a reaction without any template DNA (non-template control). All tumour and control specimens were measured in triplicates.