Pyromark q96 id

Genomic DNA was extracted by standard methods using the Wizard Genomic DNA Purification System (Promega). Bisulfite conversion of genomic DNA was carried out using the EZ DNA Methylation Kit (Zymo Research). The DNA methylation status of PRAC was assessed by PSQ using PyroMark Q96 ID (Qiagen, Valencia, CA). The primer sequences and amplification conditions are described in Table 2. PCR reactions were conducted using 20 ng of bisulfite-converted genomic DNA. A biotin-labeled primer was used to purify the final PCR product using streptavidin-coated Sepharose beads (GE Healthcare, Buckinghamshire, UK). The PCR product was bound to Sepharose beads, purified, washed, denatured using a 0.2 mol/L NaOH solution, and washed again. Subsequently, 0.3 μmol/L PSQ sequencing primer was annealed to the purified single-stranded PCR product and PSQ was performed on a PyroMark Q96 ID (Qiagen, Valencia, CA). Target CpG sites were evaluated using the instrument software (PSQ96MA 2.1, Qiagen, Valencia, CA), which converts programs to numerical values for peak heights and calculates the proportion of methylation at each base as a C/T ratio. Data analysis was performed using PyroMark Q96 ID Software v.1.0 software (Qiagen, Valencia, CA).

Pyrosequencing was used to technically validate the DNA methylation results from the Infinium HumanMethylation450 BeadChip (Illumina) in a subset of our cohort. First, DNA from human liver of 36 donors (22 males and 14 females) was bisulfite converted with the EpiTect Bisulfite Kit (Qiagen, Hilden, Germany) and then amplified with the PyroMark PCR kit. Second, pyrosequencing was performed with the PyroMark ID Q96 and PyroMark Gold Q96 reagents (Qiagen), and the PyroMark Q96 2.5.8 software program was used to analyze the DNA methylation data. Predesigned pyrosequencing assays (PCR primers and sequencing primer) (Supplemental Table 1) from Qiagen were used for the two selected CpG sites (cg05688478 annotated to APLN and cg27483305 annotated to NKAP).

Pyrosequencing analysis was performed to evaluate the frequency of MDR1 promoter methylation according to a previous study [51 (link)]. Each diagnosis was confirmed, and pyrosequencing was performed as previously described [43 (link)]. Briefly, bisulfite conversion of genomic DNA was performed using an EZ DNA Methylation-Gold Kit (Zymo Research Corporation, Irvine, CA, USA). The forward (5′ GGTTTGGGTTTTTTGGAGT 3′) and reverse (5′ CCTCCTAAAACTCCAACCT 3′) primers of MDR1 CpG island (Gene ID: 403879) were amplified by PCR (HotStarTaq Master Mix kit, Qiagen) and pyrosequencing was performed using a sequencing primer (5′ TATATTTTGGTGTTTTTG 3′) following the manufacturer’s instructions (PyroMark ID Q96, Qiagen and Biotage, Uppsala, Sweden).