Cpgenome universal methylated dna

mPDCD1 was determined by means of a methylation-specific real-time PCR assay targeting the PDCD1 promoter region [19 (link)]. The methylation-specific real-time PCR assay was duplexed with a second assay targeting a CpG-free region within the ACTB gene locus and allowing for the quantification of the total DNA, irrespective of its methylation [19 (link)]. PCR conditions (buffers, temperature cycling program, real-time PCR instrument) were applied as previously described [31 (link)]. The following primers and probes were used: PDCD1 forward primer, 5′-tcgaagcgaggttagaaatcgtt-3′; PDCD1 reverse primer, 5′-ccttcaaaaccgaaccgaatat-3′; PDCD1 probe, 5′-6-FAM-ttggcgcggttgtttggtttcgaga-BHQ-1-3′; ACTB forward primer, 5′-cccttaaaaattacaaaaaccacaa-3′; ACTB reverse primer, 5′-ggaggaggtttagtaagttttttg-3′; ACTB probe, 5′-Atto-647N-accaccacccaacacacaataacaaacaca-BHQ-2-3′. Each sample was measured in triplicate with an input of 25 ng of bisulfite-converted FFPE tissue DNA as quantified via UV. As a calibrator 3 ng of bisulfite-converted artificially methylated DNA (CpGenome Universal Methylated DNA, Merck Millipore, Billerica, MA, USA) was used. mPDCD1 was calculated with the ΔΔCT method as described earlier [31 (link)]).

For methylation analysis, samples were processed according to the instruction of InnuCONVERT Bisulfite All-In-One Kit (Analytik Jena). For assay validation, a dilution series of bisulfite-converted, unmethylated sperm DNA (NW Andrology& Cryobank Inc., Spokane, WA, USA) and artificially methylated DNA (CpGenome^™ Universal Methylated DNA; Merck Millipore, Darmstadt, Germany) were used. DNA concentration was quantified by UV spectrophotometry using a Nanodrop ND-1000 spectralphotometer (Nanodrop Technologies, Wilmington, DE, USA). The promoter region of PD-L1 was PCR amplified, T-A cloned and sequenced. Methylation analysis was performed by calculating the ratio of methylated CpG sites to total CpG sites.

Multiplex MethyLight, a methylation-specific qPCR assay was used to determine the methylation levels of APC, GSTP1, HOXD3, KLK10, TBX15, and TGFβ2 [32 (link)]. ALU-C4 (ALU) was used as a methylation-independent, sodium bisulfite conversion-dependent internal input DNA control.
Primer/probe concentrations, cycling parameters, and data acquisition/analysis were as previously described, using Applied Biosystems 7500 (Life Technologies) [12 (link)].
Gene methylation was scored as percent methylated of reference (PMR) according to Eads et al. [33 (link)] CpGenome Universal Methylated DNA (EMD Millipore) was used as the positive control and to generate standard curves. Quality control criteria included genes of interest (GOIs) standard curve R² > 0.95, ALU R² > 0.99, and slope range from − 3.28 to − 4.86. Any sample with a higher cycling threshold (lower quantity) for ALU than the least concentrated standard curve point for which all GOI amplified was excluded from analysis. Samples were analyzed in duplicate and were reanalyzed if replicates had a difference in PMR of > 10%. Data development and analysis were carried out in accordance with the Minimum Information for Publication of Quantitative real-time PCR Experiments (MIQE) guideline [34 (link)].

DNA extraction from FFPE sections was performed using FFPE RNA/DNA Purification Plus Kit (Norgen Biotek, Thorold, Canada) following the manufacturer’s instructions. DNA concentrations and purity ratios were determined using the NanoDrop Lite spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and modified with sodium bisulfite, using the EZ DNA Methylation-Gold™ Kit (Zymo Research, Orange, CA, USA) according to manufacturer’s instructions. For quantitative methylation-specific PCR (QMSP), modified DNA was used as template. Primers to specifically amplify methylated bisulfite converted complementary sequences were used and are listed in Additional Table 2. QMSP reactions were carried out in LightCyler 480 II (Roche, Germany) using 2 μL of modified DNA and 5 μL Xpert Fast SYBR (2X) (GRiSP, Porto, Portugal). All samples were run in triplicate and melting curves were obtained for each case by gene. β-actin (ACTβ) was used to normalize for DNA input in each sample [20 (link)]. To ascertain PCR efficiency, and samples’ quantification, modified CpGenome™ Universal Methylated DNA (Merck Millipore, France) was used in each plate to generate a standard curve. The relative methylation for each gene was calculated by the ratio of mean quantity for the target gene and the mean quantity of ACTβ, multiplied by 1000 for easier tabulation.

qMSP assays were designed using Beacon Designer (Premier Biosoft, Palo Alto, CA, USA). Primer and probe sequences are provided in Table S3. qMSP reactions were run in triplicates using 5 μL Taqman Universal Mastermix no UNG (Applied Biosystems, Waltham, MA, USA), 5 ng bisulfite-converted DNA, 5–12 pmol of each primer, and 2–4 pmol probe in a total volume of 10 µL. On each plate, seven-point serially diluted methylated DNA (bisulfite converted CpGenome Universal Methylated DNA (Merck Millipore, Burlington, MA, USA)) and two negative controls (H₂O and whole-genome amplified (WGA) DNA) were included. ALUC4 and MYOD1 were used for quality control and normalization (ALUC4). Reactions were run on the ViiA7 Real-Time PCR System (Applied Biosystems) in 384-well plates: 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 1 min at 56–60 °C (Table S3). Quantities were estimated from the standard curves using QuantStudio™ Real-Time PCR Software (Applied Biosystems). Outliers (>2 Ct (cycle threshold) values lower or higher than the other replicates) and samples with ALUC4 Ct > 24.0 in ≥ 2 of 3 replicate reactions and/or without a MYOD1 positive signal were removed. Samples were considered negative for methylation if ≥2 methylation-specific reactions did not amplify.

For methylation analysis, ectomy samples were processed according to the InnuConvert Bisulfite All-In-One Kit (Analytik Jena, Germany) as previously published [31 (link)]. Bisulfite DNA from biopsies were prepared as described earlier [23 ]. Length of tumor infiltrate in one tissue biopsy ranged from 0.1 mm to 12 mm (median 4 mm). Number of tumorous cores in one set of biopsies ranged from 1–8 (median 4). Tumorous tissue was marked, micro-dissected, and further processed as described above. For comparison with methylation results obtained from matched biopsies, total tumorous tissue from radical prostatectomy specimens was micro-dissected and bisulfite converted.
For the analytical performance verification of the assay, a dilution series of bisulfite-converted artificially methylated DNA (CpGenome™ Universal Methylated DNA; Merck Millipore, Darmstadt, Germany) and unmethylated DNA (NW Andrology & Cryobank Inc., Spokane, WA, USA) was used.