Methylation-specific high-resolution melting analysis (MS-HRM)-based DNA promoter profiling in paired FFPE bladder cancer samples.

2012 ◽  
Vol 30 (5_suppl) ◽  
pp. 315-315
Author(s):  
Wei Meng ◽  
Arnab Chakravarti ◽  
Tim Lautenschlaeger
Keyword(s):  
Dna Methylation ◽  
Bladder Cancer ◽  
High Resolution ◽  
Promoter Methylation ◽  
High Resolution Melting ◽  
Tissue Samples ◽  
Adjacent Tissue ◽  
Normal Adjacent Tissue ◽  
Platinum Based Chemotherapy ◽  
Cancer Tissues

315 Background: DNA methylation and histone modification are widely studied epigenetic events that can decrease gene expression levels. Promoter hypermethylation has been proposed as a potential diagnostic or prognostic biomarker in various cancers. We studied DNA promoter methylation of 5 cancer-associated genes (MRE11, APX1, ERCC1, RASSF1A and RASSF2A ) in paired FFPE bladder cancer tissues and normal adjacent tissue. The MRE11/Rad50/NBS1 complex serves as a single-strand DNA nuclease which participates in the repair of DNA double-strand breaks and replication errors. APX1 plays a key role in regulating H2O2 levels and H2O2 signaling. DNA repair machinery, ERCC1 protein levels in tumor tissue have been shown to predict response to platinum-based chemotherapy. RASSF1 is thought to be a tumor suppressor gene, while the function of RASSF2 is less well understood. Methods: 16 bladder cancer cases with available paraffin-embedded tumor and matched normal adjacent tissue specimens (32 tissue samples) were analyzed. DNA was extracted by Ambion RecoverAll Total Nucleic Acid Isolation kit. FFPE DNA bisulfite modification was performed using Zymo EZ DNA Methylation Kit. Methylation Specific-High Resolution Melting (MS-HRM) analysis was used to assess methylation status. MS-HRM monitors the melting behavior of PCR amplicons by using a DNA intercalating fluorescent dye. Results: MRE11 and APX1 promoters were not methylated in either cancer or normal adjacent samples. The ERCC1 gene was heavily methylated in 94% (30/32) of all cancer and wild type samples. RASSF1A and RASSF2A promoter methylation was significantly different between cancer and normal adjacent tissue. 50% (8/16) RASSF1A and 25% (4/16) RASSF2A had promoter methylation in cancer tissues, while only 6% (1/16) RASSF1A and 0% (0/16) RASSF2A had promoter methylation in normal adjacent tissues. 69% (11/16) of bladder cancer tissues were positive for RASSF1A or RASSF2A promotor methylation while only 1/16 normal adjacent tissue samples was positive for either promotor methylation. Conclusions: The results show that cancer and non-cancer tissue have different RASSF1A and RASSF2A promoter methylation patterns.

scholarly journals A simple and cost-effective approach for technical validation of next generation methylation sequencing data

2019 ◽  
Author(s):  
Ali Javadmanesh ◽  
Afsaneh Mojtabanezhad Shariatpanahi ◽  
Ehsan Shams Davodly ◽  
Marjan Azghandi ◽  
Maryam Yassi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
High Resolution ◽  
High Resolution Melting ◽  
Cost Effective ◽  
Next Generation ◽  
Sequencing Data ◽  
Cost Effective Approach ◽  
Technical Validation ◽  
R Programming ◽  
And Control

Abstract Background DNA methylation is a fundamental epigenetic process that, in most cases, modulates genetic expression levels. Changes in DNA methylation, either hypo- or hypermethylation, have a key role in many biological processes and several human diseases such as cancer. In the current study, we offered an approach to validate the next generation methylation sequencing data.Methods Genomic DNA was extracted from target and control samples (6 in each group), followed by bisulfite conversion. Next generation methylation sequencing and methylation sensitive high-resolution melting assay were carried out. The primers for methylation sequencing validation were designed by R programming language.Results In the current study, two groups, case and control, were discriminated based on methylation sequencing results and the real time PCR-based results were in accordance with the next generation methylation sequencing.Discussion Methylation sensitive high-resolution melting validation assay is a simple and cost-effective method, which confirmed next generation methylation sequencing results.

Assessment of PDGFRβ promoter methylation in canine osteosarcoma using methylation‐sensitive high‐resolution melting analysis

2020 ◽  
Vol 18 (4) ◽  
pp. 484-493
Author(s):  
Fabio Gentilini ◽  
Ombretta Capitani ◽  
Debora Tinto ◽  
Antonella Rigillo ◽  
Silvia Sabattini ◽  
...  
Keyword(s):  
High Resolution ◽  
Promoter Methylation ◽  
High Resolution Melting ◽  
High Resolution Melting Analysis ◽  
Canine Osteosarcoma ◽  
Melting Analysis

C51 ANALYSIS OF P16 AND DAPK PROMOTER METHYLATION IN HUMAN BLADDER CANCER TISSUES

2010 ◽  
Vol 9 (6) ◽  
pp. 632
Author(s):  
Z. Jablonowski ◽  
E. Reszka ◽  
E. Wieczorek ◽  
J. Gromadzihska ◽  
W. Wasowicz ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Promoter Methylation ◽  
Human Bladder Cancer ◽  
Human Bladder ◽  
Cancer Tissues

scholarly journals Gene Expression, DNA Methylation and Prognostic Significance of DNA Repair Genes in Human Bladder Cancer

2017 ◽  
Vol 42 (6) ◽  
pp. 2404-2417 ◽  
Author(s):  
Anita Wojtczyk-Miaskowska ◽  
Malgorzata Presler ◽  
Jerzy Michajlowski ◽  
Marcin Matuszewski ◽  
Beata Schlichtholz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Bladder Cancer ◽  
Dna Repair ◽  
Mrna Expression ◽  
Promoter Methylation ◽  
Methylation Status ◽  
Prognostic Significance ◽  
Muscle Invasive ◽  
Repair Genes

Background/Aims: This study investigated the gene expression and DNA methylation of selected DNA repair genes (MBD4, TDG, MLH1, MLH3) and DNMT1 in human bladder cancer in the context of pathophysiological and prognostic significance. Methods: To determine the relationship between the gene expression pattern, global methylation and promoter methylation status, we performed real-time PCR to quantify the mRNA of selected genes in 50 samples of bladder cancer and adjacent non-cancerous tissue. The methylation status was analyzed by methylation-specific polymerase chain reaction (MSP) or digestion of genomic DNA with a methylation-sensitive restriction enzyme and PCR with gene-specific primers (MSRE-PCR). The global DNA methylation level was measured using the antibody-based 5-mC detection method. Results: The relative levels of mRNA for MBD4, MLH3, and MLH1 were decreased in 28% (14/50), 34% (17/50) and 36% (18/50) of tumor samples, respectively. The MBD4 mRNA expression was decreased in 46% of non-muscle invasive tumors (Ta/T1) compared with 11% found in muscle invasive tumors (T2-T4) (P<0.003). Analysis of mRNA expression for TDG did not show any significant differences between Ta/T1 and T2-T4 tumors. The frequency of increased DNMT1 mRNA expression was higher in T2-T4 (52%) comparing to Ta/T1 (16%). The overall methylation rates in tumor tissue were 18% for MBD4, 25% for MLH1 and there was no evidence of MLH3 promoter methylation. High grade tumors had significantly lower levels of global DNA methylation (P=0.04). There was a significant association between shorter survival and increased expression of DNMT1 mRNA (P=0.002), decreased expression of MLH1 mRNA (P=0.032) and the presence of MLH1 promoter methylation (P=0.006). Conclusion: This study highlights the importance of DNA repair pathways and provides the first evidence of the role of MBD4 and MLH3 in bladder cancer. In addition, our findings suggest that DNMT1 mRNA and MLH1 mRNA expression, as well as the status of MLH1 promoter methylation, are attractive prognostic markers in this pathology.

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