Journal: International Journal of Molecular Sciences

Article Title: Impact of Heterogeneous DNA Methylation on the Accuracy of Quantitative Methylation-Specific PCR for Detecting DNA Hypermethylation in Prostate Cancer

doi: 10.3390/ijms27052249

Figure Lengend Snippet: Unique GSTP1 methylation patterns detected using methylation sequencing of gDNA from BPH tissue and from PCa tissue and qMSP results. ( A ) Methylation sequencing results of gDNA from BPH tissue. Each row represents a unique methylation pattern observed for the GSTP1 target region, with filled circles showing methylated CpG sites and unfilled circles showing unmethylated CpG sites. The bar plot on the left indicates the number of reads per pattern. The BPH tissue showed a majority of unmethylated fragments. ( B ) Methylation sequencing results of gDNA from PCa tissue. In contrast to BPH, PCa tissue shows a high proportion of fully methylated GSTP1 fragments, next to partly methylated fragments. ( C ) qMSP amplification curves for GSTP1 corresponding to the BPH and PCa tissue samples shown in panels A and B, together with a standard curve for reference. The BPH sample shows no amplification detected. The PCa sample, shows a clear and well-shaped amplification curve, comparable to the standard curve. : methylated CpG site, : unmethylated CpG site. BPH: benign prostate hyperplasia, PCa: prostate cancer. qMSP: Quantitative Methylation-Specific qPCR.

Article Snippet: The reaction mixture consisted of 2 μL (BT-DNA from tissue) or 10 μL (BT-DNA from urine) of bisulfite-treated gDNA, 0.4 μM of each primer, 0.1 μM of each probe, 4 μL of 5× concentrated PerfeCTa MultiPlex qPCR Toughmix (cat. no. 95147, Quantabio, Beverly, MA, USA), and nuclease-free water.

Techniques: Methylation, Sequencing, Amplification

Journal: International Journal of Molecular Sciences

Article Title: Impact of Heterogeneous DNA Methylation on the Accuracy of Quantitative Methylation-Specific PCR for Detecting DNA Hypermethylation in Prostate Cancer

doi: 10.3390/ijms27052249

Figure Lengend Snippet: Unique GSTP1 methylation patterns detected using methylation sequencing of gDNA from PCa urine and qMSP results. ( A – E ) Methylation sequencing results of gDNA isolated from urine samples of five prostate cancer patients (samples 1–5). Each row shows a unique methylation pattern observed across the GSTP1 target region, with filled circles indicating methylated CpG sites and unfilled circles indicating unmethylated CpG sites. The bar plot on the left shows the number of reads per pattern. Sample 2 (panel B ) shows the highest amount of fully methylated fragments, followed by sample 1. Both samples show, next to the presence of fully methylated fragments, also partly methylated fragments and many fully unmethylated fragments. Samples 3–5 show no or almost no fully methylated fragments, some partly methylated fragments, and many unmethylated fragments. ( F ) qMSP amplification curves for GSTP1 corresponding to samples 1–5, shown together with a standard curve for reference. Sample 1 and 2 show a clear and well-shaped amplification curve. This is consistent with the presence of fully methylated GSTP1 fragments observed via methylation sequencing (panels A , B ). In contrast, samples 3–5 show poorly shaped amplification curves, corresponding to the absence of fully methylated fragments in the sequencing data (panel C – E ). : methylated CpG site, : unmethylated CpG site. qMSP: Quantitative Methylation-Specific qPCR.

Article Snippet: The reaction mixture consisted of 2 μL (BT-DNA from tissue) or 10 μL (BT-DNA from urine) of bisulfite-treated gDNA, 0.4 μM of each primer, 0.1 μM of each probe, 4 μL of 5× concentrated PerfeCTa MultiPlex qPCR Toughmix (cat. no. 95147, Quantabio, Beverly, MA, USA), and nuclease-free water.

Techniques: Methylation, Sequencing, Isolation, Amplification