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Bisulfite Conversion of DNA from Tissues, Cell Lines, Buffy Coat, FFPE Tissues, Microdissected Cells, Swabs, Sputum, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

  • Maria Jung
  • Barbara Uhl
  • Glen Kristiansen
  • Dimo DietrichEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1589)

Abstract

Locus-specific analyses of DNA methylation patterns usually require a bisulfite conversion of the DNA, where cytosines are deaminated to uracils, while methylated and hydroxymethylated cytosines remain unaffected. The specific discrimination of hydroxymethylation and methylation can be achieved by introducing an oxidation of 5-hydroxymethylcytosines to 5-formylcytosines and subsequent bisulfite-mediated deamination of 5-formylcytosines.

DNA methylation analysis of cell-free circulating DNA in liquid biopsies, i.e., blood samples (serum and plasma), urine, aspirates, bronchial lavages, pleural effusions, and ascites, is of great interest in clinical research. However, due to the generally low concentration of circulating cell-free DNA in body fluids, high volumes need to be analyzed. A reduction of this volume, e.g., by means of a polymer-mediated enrichment, is required in order to facilitate the bisulfite conversion. Further, these sample types usually contain a cellular fraction which is of additional interest and requires specific protocols for the sample preparation.

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly used source for tissue-based clinical research. Due to degradation and covalent modifications of DNA in FFPE tissue samples, optimized protocols for the DNA preparation and bisulfite conversion are required.

This chapter describes methods and protocols for the sample preparation and subsequent high-speed bisulfite conversion and DNA clean-up for several types of relevant samples, i.e., serum, plasma, urine, buffy coat, aspirates, sputum, lavages, effusions, ascites, swabs, fresh tissues, cell lines, FFPE tissues, and laser microdissected cells.

Additionally, two real-time PCR assays for DNA quantification and quality control are described. The cytosine-free fragment (CFF) assay allows for the simultaneous quantification of bisulfite converted and total DNA and thus the determination of bisulfite conversion efficiency. The Mer9 real-time PCR assay amplifies the bisulfite converted sequence of the repetitive element Mer9 and enables the accurate quantification of minute DNA amounts, as present in microdissected cells and body fluids.

Keywords:

DNA methylation DNA hydroxymethylation Bisulfite conversion Body fluids Plasma Serum FFPE tissue Effusions Polymer-mediated enrichment DNA quantification 

Notes

Competing Interests

Dimo Dietrich has been an employee and is a stockholder of Epigenomics AG, a company that aims to commercialize the DNA methylation biomarkers SEPT9 and SHOX2. Dimo Dietrich is co-inventor and owns patents on methylation biomarkers and related technologies. These patents are commercially exploited by Epigenomics AG. Dimo Dietrich receives inventor’s compensation from Epigenomics AG. Dimo Dietrich is a consultant for AJ Innuscreen GmbH (Berlin, Germany), a 100 % daughter company of Analytik Jena AG (Jena, Germany), and receives royalties from the sale of innuCONVERT Bisulfite Kits.

References

  1. 1.
    Guibert S, Weber M (2013) Functions of DNA methylation and hydroxymethylation in mammalian development. Curr Top Dev Biol 104:47–83CrossRefPubMedGoogle Scholar
  2. 2.
    Sarkar S, Horn G, Moulton K et al (2013) Cancer development, progression, and therapy: an epigenetic overview. Int J Mol Sci 14:21087–21113CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Frommer M, McDonald LE, Millar DS et al (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A 89:1827–1831CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Darst RP, Pardo CE, Ai L et al (2010) Bisulfite sequencing of DNA. Curr Protoc Mol Biol 15:1–17, Chapter 7:Unit 7.9.1-17Google Scholar
  5. 5.
    Millar DS, Warnecke PM, Melki JR et al (2002) Methylation sequencing from limiting DNA: embryonic, fixed, and microdissected cells. Methods 27:108–113CrossRefPubMedGoogle Scholar
  6. 6.
    Boyd VL, Zon G (2004) Bisulfite conversion of genomic DNA for methylation analysis: protocol simplification with higher recovery applicable to limited samples and increased throughput. Anal Biochem 326:278–280CrossRefGoogle Scholar
  7. 7.
    Hayatsu H, Negishi K, Shiraishi M (2004) Accelerated bisulfite-deamination of cytosine in the genomic sequencing procedure for DNA methylation analysis. Nucleic Acids Symp Ser (Oxf) 48:261–262CrossRefGoogle Scholar
  8. 8.
    Hayatsu H, Shiraishi M, Negishi K (2008) Bisulfite modification for analysis of DNA methylation. Curr Protoc Nucleic Acid Chem. Chapter 6:Unit 6.10Google Scholar
  9. 9.
    Shiraishi M, Hayatsu H (2004) High-speed conversion of cytosine to uracil in bisulfite genomic sequencing analysis of DNA methylation. DNA Res 11:409–415CrossRefPubMedGoogle Scholar
  10. 10.
    Raizis AM, Schmitt F, Jost JP (1995) A bisulfite method of 5-methylcytosine mapping that minimizes template degradation. Anal Biochem 226:161–166CrossRefPubMedGoogle Scholar
  11. 11.
    Grunau C, Clark SJ, Rosenthal A (2001) Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 29:E65–65CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Tanaka K, Okamoto A (2007) Degradation of DNA by bisulfite treatment. Bioorg Med Chem Lett 17:1912–1915CrossRefPubMedGoogle Scholar
  13. 13.
    Hayatsu H (2008) The bisulfite genomic sequencing used in the analysis of epigenetic states, a technique in the emerging environmental genotoxicology research. Mutat Res 659:77–82CrossRefPubMedGoogle Scholar
  14. 14.
    Jin L, Wang W, Hu D (2013) The conversion of protonated cytosine-SO3(-) to uracil-SO3(-): insights into the novel induced hydrolytic deamination through bisulfite catalysis. Phys Chem Chem Phys 15:9034–9042CrossRefPubMedGoogle Scholar
  15. 15.
    Holmes EE, Jung M, Meller S et al (2014) Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine. PLoS One 9:e93933CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Genereux DP, Johnson WC, Burden AF et al (2008) Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies. Nucleic Acids Res 36:e150CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Esteller M, Garcia-Foncillas J, Andion E et al (2000) Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 343:1350–1354CrossRefPubMedGoogle Scholar
  18. 18.
    Stewart GD, Van Neste L, Delvenne P et al (2013) Clinical utility of an epigenetic assay to detect occult prostate cancer in histopathologically negative biopsies: results of the MATLOC study. J Urol 189:1110–1116CrossRefPubMedGoogle Scholar
  19. 19.
    Bañez LL, Sun L, van Leenders GJ et al (2010) Multicenter clinical validation of PITX2 methylation as a prostate specific antigen recurrence predictor in patients with post-radical prostatectomy prostate cancer. J Urol 184:149–156CrossRefPubMedGoogle Scholar
  20. 20.
    Dietrich D, Hasinger O, Bañez LL et al (2013) Development and clinical validation of a real-time PCR assay for PITX2 DNA methylation to predict prostate-specific antigen recurrence in prostate cancer patients following radical prostatectomy. J Mol Diagn 15:270–279CrossRefPubMedGoogle Scholar
  21. 21.
    Weiss G, Cottrell S, Distler J et al (2009) DNA methylation of the PITX2 gene promoter region is a strong independent prognostic marker of biochemical recurrence in patients with prostate cancer after radical prostatectomy. J Urol 181:1678–1685CrossRefPubMedGoogle Scholar
  22. 22.
    Schatz P, Dietrich D, Koenig T et al (2010) Development of a diagnostic microarray assay to assess the risk of recurrence of prostate cancer based on PITX2 DNA methylation. J Mol Diagn 12:345–353CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Weller M, Stupp R, Reifenberger G et al (2010) MGMT promoter methylation in malignant gliomas: ready for personalized medicine? Nat Rev Neurol 6:39–51CrossRefPubMedGoogle Scholar
  24. 24.
    Blow N (2007) Tissue preparation: tissue issues. Nature 448:959–963CrossRefPubMedGoogle Scholar
  25. 25.
    Bereczki L, Kis G, Bagdi E et al (2007) Optimization of PCR amplification for B- and T-cell clonality analysis on formalin-fixed and paraffin-embedded samples. Pathol Oncol Res 13:209–214CrossRefPubMedGoogle Scholar
  26. 26.
    Dietrich D, Uhl B, Sailer V et al (2013) Improved PCR performance using template DNA from formalin-fixed and paraffin-embedded tissues by overcoming PCR inhibition. PLoS One 8:e77771CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Kuykendall JR, Bogdanffy MS (1992) Efficiency of DNA-histone crosslinking induced by saturated and unsaturated aldehydes in vitro. Mutat Res 283:131–136CrossRefPubMedGoogle Scholar
  28. 28.
    Church TR, Wandell M, Lofton-Day C et al (2014) Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer. Gut 63:317–325CrossRefPubMedGoogle Scholar
  29. 29.
    deVos T, Tetzner R, Model F et al (2009) Circulating methylated SEPT9 DNA in plasma is a biomarker for colorectal cancer. Clin Chem 55:1337–1346CrossRefPubMedGoogle Scholar
  30. 30.
    Kneip C, Schmidt B, Seegebarth A et al (2011) SHOX2 DNA methylation is a biomarker for the diagnosis of lung cancer in plasma. J Thorac Oncol 6:1632–1638CrossRefPubMedGoogle Scholar
  31. 31.
    Dietrich D, Jung M, Puetzer S et al (2013) Diagnostic and prognostic value of SHOX2 and SEPT9 DNA methylation and cytology in benign, paramalignant and malignant pleural effusions. PLoS One 8:e84225CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Jung J, Kristiansen G, Dietrich D (2015) DNA methylation analysis of free-circulating DNA in body fluids. Methods Mol Biol. In press.Google Scholar
  33. 33.
    Booth MJ, Ost TW, Beraldi D et al (2013) Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat Protoc 8:1841–1851CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Booth MJ, Balasubramanian S (2014) Methods for detection of nucleotide modification. US Patent 14/235,707, 26 June 2014Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Maria Jung
    • 1
  • Barbara Uhl
    • 1
  • Glen Kristiansen
    • 1
  • Dimo Dietrich
    • 1
    Email author
  1. 1.Institute of PathologyUniversity Hospital Bonn (UKB)BonnGermany

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