Journal: The World Journal of Men's Health

Article Title: A Highly Sensitive Methylation Assay for Prostate Cancer Diagnosis

doi: 10.5534/wjmh.240182

Figure Lengend Snippet: The Tri-Component Liquid Biopsy Assay for Optimized DNA Methylation Detection. Unmethylated cytosines changed to uracil after bisulfite treatment, while methylated ones remained constant. In the PCR session, a blocker strand was added to the methylation-specific PCR, forming a three-stranded DNA structure with the template and primers that allows for enrichment of methylated DNA. The blocker strand is fully complementary to the wild type (WT) DNA template and can hinder polymerase amplification, while its mismatch with the methylated DNA template (MT) hardly affects the amplification process. Afterwards, a fluorescent probe with an AP site was used to specifically recognize the methylation site with the assistance of endonuclease IV. AP: apurinic/apyrimidinic.

Article Snippet: Additionally, the EZ DNA Methylation Bisulfite Conversion Kit was acquired from Zymo Research.

Techniques: DNA Methylation Assay, Methylation, Amplification

Journal: The World Journal of Men's Health

Article Title: A Highly Sensitive Methylation Assay for Prostate Cancer Diagnosis

doi: 10.5534/wjmh.240182

Figure Lengend Snippet: Feasibility studies of Tri-Component Liquid Biopsy Assay. (A, B) Efficacy of the guided strand-assisted detection system in discriminating between different abundances of methylated DNA strands. (C, D) Methylation-specific PCR (MSP) alone can only enhance mutation abundance by about 100-fold (C), whereas blocker-assisted MSP can increase mutation abundance by at least 5000-fold (D). (E) The detection limit of the PCR enrichment and guide strand-assisted enzyme/probe detection system can be as low as 0.002% for methylated standard samples. WT: wild type.

Article Snippet: Additionally, the EZ DNA Methylation Bisulfite Conversion Kit was acquired from Zymo Research.

Techniques: Methylation, Mutagenesis

Journal: The World Journal of Men's Health

Article Title: A Highly Sensitive Methylation Assay for Prostate Cancer Diagnosis

doi: 10.5534/wjmh.240182

Figure Lengend Snippet: The efficacy of the assay in clinical samples. (A) Procedures for methylation testing of samples in the clinic. (B) Distribution of DNA methylation abundance at ADCY4, AOX1, GSTP1, HOXA7, RASSF5, and SFRP5 sites in urine samples from asymptomatic control (AC, n=17) and prostate cancer (PCa, n=24). Red areas indicate below the detection limit of the assay and fluorescence intensity is indistinguishable from wild type (WT). (C) ROC curves were plotted and area under curve (AUC) was calculated based on the results of DNA methylation levels of ADCY4, AOX1, GSTP1, HOXA7, RASSF5, and SFRP5 sites detected by DNA Methylation Assay Platform, respectively. (D) Percentage of cases determined by testing urine samples from PCa patients and blood samples from ACs with the DNA methylation analysis platform.

Article Snippet: Additionally, the EZ DNA Methylation Bisulfite Conversion Kit was acquired from Zymo Research.

Techniques: Methylation, DNA Methylation Assay, Control, Fluorescence

Journal: The World Journal of Men's Health

Article Title: A Highly Sensitive Methylation Assay for Prostate Cancer Diagnosis

doi: 10.5534/wjmh.240182

Figure Lengend Snippet: A panel of methylation sites for PCa diagnosis. (A) Distribution of DNA methylation abundance at the GSTP1, ADCY4, and HOXA7 sites in plasma free DNA from ACs (n=17) and PCa (n=55). Red areas indicate below the detection limit of the assay and fluorescence intensity is indistinguishable from WT. (B) ROC curve of the 3-gene detection panel. (C) PCa patients and healthy population controls for plasma free DNA methylation variability were determined with a sensitivity of 89% for this method. (D–F) The relationship between methylation patterns and Gleason score (D), PSA level (E), and PI-RADS score (F). PCa: prostate cancer, AC: asymptomatic control, WT: wild type, PSA: prostate-specific antigen, PI-RADS: prostate imaging-reporting and data system. Statistically significant ( * p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Additionally, the EZ DNA Methylation Bisulfite Conversion Kit was acquired from Zymo Research.

Techniques: Methylation, Biomarker Discovery, DNA Methylation Assay, Clinical Proteomics, Fluorescence, Control, Imaging