Abstract
The defining feature of the Quantitative Multiplex Methylation-Specific PCR (QM-MSP) method to sensitively quantify DNA methylation is the two-step PCR approach for a multiplexed analysis of a panel of up to 12 genes in clinical samples with minimal quantities of DNA. In the first step, for up to 12 genes tested, one pair of gene-specific primers (forward and reverse) amplifies the methylated and unmethylated copies of the same gene simultaneously and in multiplex, in one PCR reaction. This methylation-independent amplification step produces amplicons of up to 109 copies per μL after 36 cycles of PCR. In the second step, the amplicons of the first reaction (STEP 1) are quantified with a standard curve using real-time PCR and two independent fluorophores to detect methylated/unmethylated DNA of each gene in the same well (e.g., 6FAM and VIC). One methylated copy is detectable in 100,000 reference gene copies. Methylation is reported on a continuous scale. For the gene panel, the highest level of normal DNA methylation above which a sample would be called positive is derived by using Receiver Operating Characteristic (ROC), maximizing assay specificity and sensitivity to distinguish between normal/benign versus tumor DNA. QM-MSP can be applied to clinical samples of fresh or fixed ductal cells, ductal fluid, nipple fluid, fine needle aspirates, core biopsies, and tumor tissue sections.
This is a preview of subscription content, log in via an institution.
References
Swift-Scanlan T, Blackford A, Argani P et al (2006) Two-color quantitative multiplex methylation-specific PCR. Biotechniques 40:210–219
Fackler MJ, McVeigh M, Mehrotra J et al (2004) Quantitative multiplex methylation-specific PCR assay for the detection of promoter hypermethylation in multiple genes in breast cancer. Cancer Res 64:4442–4452
Fackler MJ, Malone K, Zhang Z et al (2006) Quantitative multiplex methylation-specific PCR analysis doubles detection of tumor cells in breast ductal fluid. Clin Cancer Res 12:3306–3310
Euhus DM, Bu D, Ashfaq R et al (2007) Atypia and DNA methylation in nipple duct lavage in relation to predicted breast cancer risk. Cancer Epidemiol Biomarkers Prev 16:1812–1821
Lee JS, Lo PK, Fackler MJ et al (2007) A comparative study of Korean with Caucasian breast cancer reveals frequency of methylation in multiple genes correlates with breast cancer in young, ER, PR-negative breast cancer in Korean women. Cancer Biol Ther 6:1114–1120
Locke I, Kote-Jarai Z, Fackler MJ et al (2007) Gene promoter hypermethylation in ductal lavage fluid from healthy BRCA gene mutation carriers and mutation-negative controls. Breast Cancer Res 9:R20
Euhus DM, Bu D, Milchgrub S et al (2008) DNA methylation in benign breast epithelium in relation to age and breast cancer risk. Cancer Epidemiol Biomarkers Prev 17:1051–1059
Suijkerbuijk KP, Fackler MJ, Sukumar S et al (2008) Methylation is less abundant in BRCA1-associated compared with sporadic breast cancer. Ann Oncol 19:1870–1874
Wu JM, Fackler MJ, Halushka MK et al (2008) Heterogeneity of breast cancer metastases: comparison of therapeutic target expression and promoter methylation between primary tumors and their multifocal metastases. Clin Cancer Res 14:1938–1946
Fackler MJ, Rivers A, Teo WW et al (2009) Hypermethylated genes as biomarkers of cancer in women with pathologic nipple discharge. Clin Cancer Res 15:3802–3811
Herman JG, Graff JR, Myohanen S et al (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93:9821–9826
Eads CA, Danenberg KD, Kawakami K et al (2000) MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 28:E32
Lehmann U, Langer F, Feist H et al (2002) Quantitative assessment of promoter hypermethylation during breast cancer development. Am J Pathol 160:605–612
Palmisano WA, Divine KK, Saccomanno G et al (2000) Predicting lung cancer by detecting aberrant promoter methylation in sputum. Cancer Res 60:5954–5958
Fackler MJ, Umbricht CB, Williams D et al (2011) Genome-wide methylation analysis identifies genes specific to breast cancer hormone receptor status and risk of recurrence. Cancer Res 71:6195–6207
Fackler MJ, Lopez Bujanda Z, Umbricht C et al (2014) Novel methylated biomarkers and a robust assay to detect circulating tumor DNA in metastatic breast cancer. Cancer Res 74:2160–2170
Acknowledgments
We thank Sidra Hafeez for critical review of the manuscript. This work was supported by grants to SS from AVON Research Foundation, the Rubenstein family, John A. Sellon Charitable Trust, the Department of Defense Center of Excellence on “Targeting Metastatic Breast Cancer” grant W81XWH-04-1-0595, and SKCCC Core grant P30 CA006973.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Fackler, M.J., Sukumar, S. (2018). Quantitation of DNA Methylation by Quantitative Multiplex Methylation-Specific PCR (QM-MSP) Assay. In: Tost, J. (eds) DNA Methylation Protocols. Methods in Molecular Biology, vol 1708. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7481-8_24
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7481-8_24
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7479-5
Online ISBN: 978-1-4939-7481-8
eBook Packages: Springer Protocols