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PMID: 16645207 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis.

The Journal of molecular diagnostics : JMD ·Vol. 8 ·No. 2 ·2006-05-00 ·Pages 209-17

Ogino S, Kawasaki T, Brahmandam M, Cantor M, Kirkner GJ, Spiegelman D, Makrigiorgos GM, Weisenberger DJ, Laird PW, Loda M, Fuchs CS

Abstract

Assays to measure DNA methylation, which are important in epigenetic research and clinical diagnostics, typically rely on conversion of unmethylated cytosine to uracil by sodium bisulfite. However, no study has comprehensively evaluated the precision and performance characteristics of sodium bisulfite conversion and subsequent quantitative methylation assay. We developed quantitative real-time polymerase chain reaction (MethyLight) to measure percentage of methylated reference (PMR, ie, degree of methylation) for the MGMT, MLH1, and CDKN2A (p16) promoters. To measure the precision of bisulfite conversion, we bisulfite-treated seven different aliquots of DNA from each of four paraffin-embedded colon cancer samples. To assess run-to-run variation, we repeated MethyLight five times. Bisulfite-to-bisulfite coefficient of variation (CV) of PMR ranged from 0.10 to 0.38 (mean, 0.21), and run-to-run CV of PMR ranged from 0.046 to 0.60 (mean, 0.31). Interclass correlation coefficients were 0.74 to 0.84 for the three loci, indicating good reproducibility. DNA mixing study with methylated and unmethylated DNA showed good linearity of the assay. Of 272 colorectal cancers evaluated, most showed PMR either <1 or >10, and promoter methylation (PMR >4) was tightly associated with loss of respective protein expression (P < 10(-16)). In conclusion, sodium bisulfite conversion and quantitative MethyLight assays have good precision and linearity and can be effectively used for high-throughput DNA methylation analysis on paraffin-embedded tissue.

MeSH Terms
Adaptor Proteins, Signal Transducing Carrier Proteins/genetics Collagen Type II/genetics Colorectal Neoplasms/genetics Cyclin-Dependent Kinase Inhibitor p16/genetics DNA/metabolism DNA Methylation Gene Expression Gene Expression Regulation, Neoplastic Humans MutL Protein Homolog 1 Nuclear Proteins/genetics Polymerase Chain Reaction/methods Sulfites/analysis Time Factors
Chemicals
Adaptor Proteins, Signal Transducing COL2A1 protein, human Carrier Proteins Collagen Type II Cyclin-Dependent Kinase Inhibitor p16 MLH1 protein, human Nuclear Proteins Sulfites DNA MutL Protein Homolog 1
Authors & Affiliations
11 authors, click to expand affiliations / ORCID
Ogino Shuji
Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115, USA. shuji_ogino@dfci.harvard.edu
Kawasaki Takako
Brahmandam Mohan
Cantor Mami
Kirkner Gregory J
Spiegelman Donna
Makrigiorgos G Mike
Weisenberger Daniel J
Laird Peter W
Loda Massimo
Fuchs Charles S
References (42)
42 references, click to expand
  1. Distinct molecular features of colorectal carcinoma with signet ring cell component and colorectal carcinoma with mucinous component.
    Mod Pathol. 2006 Jan;19(1):59-68 PMID: 16118624
  2. A real-time PCR assay for DNA-methylation using methylation-specific blockers.
    Nucleic Acids Res. 2004 Jan 13;32(1):e10 PMID: 14722226
  3. CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression.
    Cancer Res. 1999 May 15;59(10):2302-6 PMID: 10344733
  4. Methylation matters: a new spin on maspin.
    Nat Genet. 2002 Jun;31(2):123-4 PMID: 12040372
  5. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.
    Nat Genet. 2000 Feb;24(2):132-8 PMID: 10655057
  6. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.
    Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9821-6 PMID: 8790415
  7. MGMT gene silencing and benefit from temozolomide in glioblastoma.
    N Engl J Med. 2005 Mar 10;352(10):997-1003 PMID: 15758010
  8. Development and assessment of a quantitative reverse transcription-PCR assay for simultaneous measurement of four amplicons.
    Clin Chem. 2003 Sep;49(9):1467-75 PMID: 12928227
  9. Cancer epigenetics.
    Hum Mol Genet. 2005 Apr 15;14 Spec No 1:R65-76 PMID: 15809275
  10. Conversion-specific detection of DNA methylation using real-time polymerase chain reaction (ConLight-MSP) to avoid false positives.
    Methods. 2002 Jun;27(2):114-20 PMID: 12095268
  11. Analysis of methylated genes in peritoneal fluids of ovarian cancer patients: a new prognostic tool.
    Clin Chem. 2004 Nov;50(11):2171-3 PMID: 15502091
  12. The Nurses' Health Study: lifestyle and health among women.
    Nat Rev Cancer. 2005 May;5(5):388-96 PMID: 15864280
  13. PGP9.5 promoter methylation is an independent prognostic factor for esophageal squamous cell carcinoma.
    Cancer Res. 2005 Jun 1;65(11):4963-8 PMID: 15930319
  14. Gene-specific methylation and subsequent risk of colorectal adenomas among participants of the polyp prevention trial.
    Cancer Epidemiol Biomarkers Prev. 2005 May;14(5):1219-23 PMID: 15894675
  15. Quantitation of GSTP1 methylation in non-neoplastic prostatic tissue and organ-confined prostate adenocarcinoma.
    J Natl Cancer Inst. 2001 Nov 21;93(22):1747-52 PMID: 11717336
  16. Quantitation of viral DNA by real-time PCR applying duplex amplification, internal standardization, and two-color fluorescence detection.
    Appl Environ Microbiol. 2001 Jun;67(6):2837-9 PMID: 11375203
  17. Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen.
    Cancer Res. 2004 Jun 1;64(11):3807-13 PMID: 15172987
  18. CpG island methylator phenotype in cancer.
    Nat Rev Cancer. 2004 Dec;4(12):988-93 PMID: 15573120
  19. Fields of aberrant CpG island hypermethylation in Barrett's esophagus and associated adenocarcinoma.
    Cancer Res. 2000 Sep 15;60(18):5021-6 PMID: 11016622
  20. Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis.
    Electrophoresis. 2002 Dec;23(24):4072-9 PMID: 12481262
  21. Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing.
    Biotechniques. 2003 Jul;35(1):152-6 PMID: 12866415
  22. An analytical method for the detection of methylation differences at specific chromosomal loci using primer extension and ion pair reverse phase HPLC.
    Hum Mutat. 2002 Oct;20(4):305-11 PMID: 12325026
  23. Bisulfite genomic sequencing: systematic investigation of critical experimental parameters.
    Nucleic Acids Res. 2001 Jul 1;29(13):E65-5 PMID: 11433041
  24. MethyLight: a high-throughput assay to measure DNA methylation.
    Nucleic Acids Res. 2000 Apr 15;28(8):E32 PMID: 10734209
  25. Quantitative methylation analysis using methylation-sensitive single-nucleotide primer extension (Ms-SNuPE).
    Methods. 2002 Jun;27(2):128-33 PMID: 12095270
  26. COBRA: a sensitive and quantitative DNA methylation assay.
    Nucleic Acids Res. 1997 Jun 15;25(12):2532-4 PMID: 9171110
  27. Quantification of PCR bias caused by a single nucleotide polymorphism in SMN gene dosage analysis.
    J Mol Diagn. 2002 Nov;4(4):185-90 PMID: 12411585
  28. A quantitative promoter methylation profile of prostate cancer.
    Clin Cancer Res. 2004 Dec 15;10(24):8472-8 PMID: 15623627
  29. MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome.
    Nucleic Acids Res. 2004 Dec 02;32(21):e168 PMID: 15576675
  30. Epigenetic patterns in the progression of esophageal adenocarcinoma.
    Cancer Res. 2001 Apr 15;61(8):3410-8 PMID: 11309301
  31. Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology.
    Clin Chem. 2001 Apr;47(4):654-60 PMID: 11274014
  32. The power and the promise of DNA methylation markers.
    Nat Rev Cancer. 2003 Apr;3(4):253-66 PMID: 12671664
  33. Rapid analysis of CpG methylation patterns using RNase T1 cleavage and MALDI-TOF.
    Nucleic Acids Res. 2004 Dec 02;32(21):e167 PMID: 15576674
  34. Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites.
    Biotechniques. 2003 Jul;35(1):146-50 PMID: 12866414
  35. Simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography.
    Nucleic Acids Res. 2002 Feb 1;30(3):E13 PMID: 11809901
  36. Circulating tumor-specific DNA: a marker for monitoring efficacy of adjuvant therapy in cancer patients.
    Cancer Res. 2005 Feb 15;65(4):1141-5 PMID: 15734995
  37. The influence of folate and multivitamin use on the familial risk of colon cancer in women.
    Cancer Epidemiol Biomarkers Prev. 2002 Mar;11(3):227-34 PMID: 11895870
  38. A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus.
    Nucleic Acids Res. 2004 Sep 07;32(16):e125 PMID: 15353561
  39. Ligation-mediated PCR improves the sensitivity of methylation analysis by restriction enzymes and detection of specific DNA strand breaks.
    Nucleic Acids Res. 1990 Mar 25;18(6):1435-9 PMID: 2158077
  40. DNA methylation, field effects, and colorectal cancer.
    J Natl Cancer Inst. 2005 Sep 21;97(18):1317-9 PMID: 16174847
  41. CpG island methylator phenotype (CIMP) of colorectal cancer is best characterised by quantitative DNA methylation analysis and prospective cohort studies.
    Gut. 2006 Jul;55(7):1000-6 PMID: 16407376
  42. MGMT promoter methylation and field defect in sporadic colorectal cancer.
    J Natl Cancer Inst. 2005 Sep 21;97(18):1330-8 PMID: 16174854
Article Info
Journal
The Journal of molecular diagnostics : JMD
Abbr.
J Mol Diagn
ISSN
1525-1578
DOI
10.2353/jmoldx.2006.050135
Email
shuji_ogino@dfci.harvard.edu
Published
2006-05-00
Pages
209-17
Language
English
Region
United States
NLM ID
100893612
PMCID
PMC1867588
Subset
IM
Grants
NCI NIH HHS · P01 CA087969 · United States
NCI NIH HHS · R01 CA075090 · United States
NCI NIH HHS · P01 CA055075 · United States
NCI NIH HHS · P01 CA55075-13 · United States
NCI NIH HHS · P01 CA87969-03 · United States
Corrections
CommentIn
PMID: 16645200
External Links
PubMed source DOI: 10.2353/jmoldx.2006.050135 PMC Full Text View journal details
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