Skip to main content

Advertisement

SpringerLink
Log in

A methylation-specific dot blot assay for improving specificity and sensitivity of methylation-specific PCR on DNA methylation analysis

  • Original Article
  • Published:
International Journal of Clinical Oncology Aims and scope Submit manuscript

Abstract

Background and objectives

Developing a methylation-specific dot blot assay (MSP-DB) to increase the sensitivity and specificity of simultaneous methylation analysis of multiple genes is the goal of the present study to evaluate the methylation status of GSTP1 and RASSF1A from prostate cancer in Vietnamese males.

Methods

The methylation of GSTP1 and RASSF1A was investigated by using the MSP in 50 prostate cancer and 17 benign prostate hyperplasia specimens. The MSP-DB assay that uses a single or multiple probes specifically detected the methylation status of a particular gene or of the two genes GSTP1 and RASSF1A at the same time in a series of samples. The sensitivity and specificity of the MSP-DB were compared to those of the MSP.

Results

The probes specifically hybridized with the methylated targets only in the MSP-DB, which allowed detecting GSTP1 and RASSF1A methylation in 23 of 50 and 14 of 50 patients with prostate cancer and in 2 of 17 and 4 of 17 patients with benign prostate hyperplasia. MSP-DB following the MSP assay improved the sensitivity of detection to more than 0.01 % methylated status of a given high CpG-rich region. One methylated MSP product corresponding to the GSTP1, lack of methylated cytosine, was clearly detected on gel electrophoresis but barely visible on MSP-DB. Thus, the MSP-DB is suitable to eliminate the risk of false-positive results.

Conclusion

The MSP-DB dispels the weakness of MSP and increases the sensitivity to simultaneous methylation analysis of multiple genes. The MSP-DB is advantageous for the promotion of DNA methylation markers in progressing quickly toward clinical application.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Heyn H, Esteller M (2012) DNA methylation profiling in the clinic: applications and challenges. Nat Rev Genet 13:679–683

    Article  CAS  PubMed  Google Scholar 

  2. Shena L, Waterland RA (2012) Methods of DNA methylation analysis. Curr Opin Clin Nutr Metab Care 10:576–581

    Article  Google Scholar 

  3. Clark SJ, Statham A, Stirzaker C et al (2006) DNA methylation: bisulphite modification and analysis. Nat Protocol 1:2355–2365

    Google Scholar 

  4. Herman JG, Graff JR, Myohanen S (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93:9821–9826

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Lan TV, Ha TN, Uyen QN et al (2014) Standardization of the methylation-specific PCR method for analysing BRCA1 and ER methylation. Mol Med Rep 9:1844–1850

    CAS  PubMed  Google Scholar 

  6. Yamamoto N, Nakayama T, Kajita M et al (2012) Detection of aberrant promoter methylation of GSTP1, RASSF1A, and RARβ2 in serum DNA of patients with breast cancer by a newly established one-step methylation-specific PCR assay. Breast Cancer Res Treat 132:165–173

    Article  CAS  PubMed  Google Scholar 

  7. Zhang Q, Hu G, Yang Q et al (2013) A multiplex methylation-specific PCR assay for the detection of early-stage ovarian cancer using cell-free serum DNA. Gynecol Oncol 130:132–139

    Article  CAS  PubMed  Google Scholar 

  8. Castellanos-Rizaldos E, Milbury CA, Karatza E et al (2014) COLD-PCR amplification of bisulfite-converted DNA allows the enrichment and sequencing of rare unethylated genomic regions. PLoS ONE 9(4):e94103.doi:10.1371

  9. Kristensen LS, Hansen LL (2009) PCR-based methods for detecting single-locus DNA methylation biomarkers in cancer diagnostics, prognostics, and response to treatment. Clin Chem 55:1471–1483

    Article  CAS  PubMed  Google Scholar 

  10. Thomassin H, Kress C, Grange T (2004) MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome. Nucleic Acids Res 32:e168. doi:10.1093

  11. Hattermann K, Mehdorn HM, Mentlein R (2008) A methylation-specific and SYBR-green-based quantitative polymerase chain reaction technique for O6-methyl guanine DNA methyltransferase promoter methylation analysis. Anal Biochem 377:62–71

    Article  CAS  PubMed  Google Scholar 

  12. Eads CA, Danenberg KD, Kawakami K et al (2000) MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 28:e32

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Cottrell SE, Distler J, Goodman NS et al (2004) A real-time PCR assay for DNA-methylation using methylation-specific blockers. Nucleic Acids Res 32:e10

    Article  PubMed Central  PubMed  Google Scholar 

  14. Wojdacz TK, Dobrovic A (2007) Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res 35(6):e41. doi:10.1093

  15. Gitan RS, Shi H, Chen CM et al (2001) Methylation-specific oligonucleotide microarray: a new potential for high-throughput methylation analysis. Genome Res 12:158–164

    Article  Google Scholar 

  16. Clément G, Benhattar J (2005) A methylation sensitive dot blot assay (MSP-DBA) for the quantitative analysis of DNA methylation in clinical samples. J Clin Pathol 58:155–158

    Article  PubMed Central  PubMed  Google Scholar 

  17. Dimberg J, Thai TH, Skarstedt M et al (2012) Analysis of APC and IGFBP7 promoter gene methylation in Swedish and Vietnamese colorectal cancer patients. Oncol Lett 5:25–30

    PubMed Central  PubMed  Google Scholar 

  18. Lan TV, Thuan BT, Thu DM et al (2013) Methylation profile of BRCA1, RASSF1A and ER in Vietnamese women with ovarian cancer. Asian Pac J Cancer Prev 14:7713–7718

    Article  Google Scholar 

  19. Houshdaran S, Hawley S, Palmer C et al (2010) DNA methylation profiles of ovarian epithelial carcinoma tumors and cell lines.  PLoS ONE 5(2):e9359. doi:10.1371/journal.pone.0009359

    Article  PubMed Central  PubMed  Google Scholar 

  20. Feng S, Rubbi L, Jacobsen SE et al (2011) Determining DNA methylation profiles using sequencing. Methods Mol Biol 733:223–228

    Article  CAS  PubMed  Google Scholar 

  21. Warnecke PM, Stirzaker C, Melki JR et al (1997) Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Res 25:4422–4426

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Van Neste L, Bigley J, Toll A et al (2012) A tissue biopsy-based epigenetic multiplex PCR assay for prostate cancer detection. BMC Urol 12:16. doi:1471-2490/12/16

  23. Raluca D, Maria P, Dana D et al (2013) Molecular detection of prostate cancer using a panel of DNA methylation biomarkers. Eur Sci J 9:382–389

    Google Scholar 

  24. Yi JM, Guzzetta AA, Bailey VJ et al (2013) Novel methylation biomarker panel for the early detection of pancreatic cancer. Clin Cancer 19:6544–6555

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was financially supported by Grant KC.04.05/11-15 from the Ministry of Science and Technology, Viet Nam.

Conflict of interest

The authors Vo Thi Thuong Lan, Vuong Dieu Linh, Nguyen Thu Trang, Doan Thi Hong Van, Ta Van To, Nguyen Quynh Uyen and Ta Bich Thuan declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Faculty of Biology, VNU University of Science, 334, Nguyen Trai, Thanh Xuan, Hanoi, Vietnam

    Vo Thi Thuong Lan, Nguyen Thu Trang, Doan Thi Hong Van & Ta Bich Thuan

  2. Department of Cytology and Pathology, National Cancer Hospital K, 43 Quan Su, Hoan Kiem, Hanoi, Vietnam

    Ta Van To & Vuong Dieu Linh

  3. Institute of Microbiology and Biotechnology, Vietnam National University Hanoi, 144, Xuan Thuy, Cau Giay, Hanoi, Vietnam

    Nguyen Quynh Uyen

Authors
  1. Vo Thi Thuong Lan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nguyen Thu Trang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Doan Thi Hong Van
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ta Bich Thuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ta Van To
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Vuong Dieu Linh
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Nguyen Quynh Uyen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vo Thi Thuong Lan.

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lan, V.T.T., Trang, N.T., Van, D.T.H. et al. A methylation-specific dot blot assay for improving specificity and sensitivity of methylation-specific PCR on DNA methylation analysis. Int J Clin Oncol 20, 839–845 (2015). https://doi.org/10.1007/s10147-014-0780-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10147-014-0780-5

Keywords

Search

Navigation