Abstract
Methylation of cytosines in CpG dinucleotides is an important regulator of gene expression in the human genome. Aberrant methylation is frequently observed in carcinogenesis and its detection has been exploited for diagnostic purposes, for prognosis, or as a predictive tool of response to therapy. Currently, most methods for assessing methylation rely on bisulfite conversion of the DNA, which causes cytosine conversion to uracil, while leaving methylated cytosines unmodified. Since this modification is detrimental for DNA quality and could yield artifacts due to incomplete conversion, its chemistry needs to be tailored to the analyzed material. This chapter provides the protocols for: a single-locus assay relying on a nested, bisulfite modification-specific methylation-specific PCR (BM-MSP) design. protocols for bisulfite modification and DNA extraction consistent with FFPE-derived material are also supplied. a multilocus assay based on methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), which is particularly suitable for FFPE as it does not need bisulfite conversion and performs well on short sequences.
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Notes
- 1.
The solubilization of proteinase K in 50% sterile glycerol avoids the freezing effect at −20°C, maintaining an optimal enzymatic activity.
- 2.
Clean the microtome with xylene. Cool the paraffin blocks at −20°C or on dry ice in aluminum foil to obtain thin sections.
- 3.
The number of sections may vary, according to the tissue type and quantity.
- 4.
Use some sections from a paraffin block without any tissue for the negative controls.
- 5.
When working with xylene, avoid breathing fumes; it is better to perform the deparaffinization step under a chemical hood.
- 6.
Wear gloves when isolating and handling DNA to minimize the activity of endogenous nucleases.
- 7.
Discard the supernatant using a 1micropipette or a glass Pasteur pipette. Disposal of chemicals should be done in keeping with your laboratory safety rules.
- 8.
Methanol is toxic by inhalation. Work under a chemical hood.
- 9.
A 48-h digestion might be beneficial to bisulfite modification when contamination from melanin is expected. In such a case, a further Proteinase K addition is advisable after 24 h.
- 10.
We prefer metabisulfite to sodium bisulfite. The precise molarity of the latter cannot be estimated as commercially available sodium bisulfite is a mixture of sodium bisulfite and metabisulfite.
- 11.
Store in the dark. This solution is stable for weeks at room temperature. Discard if it turns yellow.
- 12.
Sodium Metabisulfite and hydroquinone powders are harmful by inhalation. Operate under a chemical hood. To dissolve metabisulfite, shake under a warm water-spout.
- 13.
Do not exceed this quantity, as the bisulfite amount is the limiting factor if 1.5 ml tubes are used. If you use ≤1 μg of DNA, add Herring DNA (yeast RNA or tRNA from Sigma, cat. R8508) to 2 μg final nucleic acid as a carrier during BM.
- 14.
Herring DNA is added at this point to enhance the binding of small quantities of DNA to the resin.
- 15.
This control can be performed only once, when the method is settled.
- 16.
As residual resin may be present in DNA suspension, spin the tubes for 2′ at 14,000 RPM before adding the DNA. Do not exceed suggested volumes, as BM DNA may contain PCR inhibitors.
- 17.
6× loading buffer: 0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol in H2O.
- 18.
The choice of the dilution relies on band signal intensity at visual inspection. If no band is visible, use 5 ml of undiluted PCR product. Prepare dilutions and PCR master mixes in different rooms. Avoid moving equipment and disposables from one room to the other.
- 19.
See Footnote 17
- 20.
Optional: purify PCR product from the agarose gel or directly with a PCR cleanup kit. In the latter case, use 40 μl of PCR product.
References
Ehrlich M, Gama-Sosa MA, Huang LH, Midgett RM, Kuo KC, McCune RA, Gehrke C (1982) Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells. Nucleic Acids Res 10(8):2709–2721
Li E (2002) Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet 3(9):662–673
Jones PA (1996) DNA methylation errors and cancer. Cancer Res 56(11):2463–2467
Jones PA, Baylin SB (2002) The fundamental role of epigenetic events in cancer. Nat Rev Genet 3(6):415–428
Robertson KD, Wolffe AP (2000) DNA methylation in health and disease. Nat Rev Genet 1(1):11–19
Laird PW (2003) The power and the promise of DNA methylation markers. Nat Rev Cancer 3(4):253–266
Fraga MF, Esteller M (2002) DNA methylation: a profile of methods and applications. Biotechniques 33(3):632–634, 636–649
Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA 89(5):1827–1831
Grunau C, Clark SJ, Rosenthal A (2001) Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 29(13):E65
Millar DS, Warnecke PM, Melki JR, Clark SJ (2002) Methylation sequencing from limiting DNA: embryonic, fixed, and microdissected cells. Methods 27(2):108–113
Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 93(18):9821–9826
Sasaki M, Anast J, Bassett W, Kawakami T, Sakuragi N, Dahiya R (2003) Bisulfite conversion-specific and methylation-specific PCR: a sensitive technique for accurate evaluation of CpG methylation. Biochem Biophys Res Commun 309(2):305–309
Nygren AO, Ameziane N, Duarte HM, Vijzelaar RN, Waisfisz Q, Hess CJ, Schouten JP, Errami A (2005) Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res 33(14):e128
Palmisano WA, Divine KK, Saccomanno G, Gilliland FD, Baylin SB, Herman JG, Belinsky SA (2000) Predicting lung cancer by detecting aberrant promoter methylation in sputum. Cancer Res 60(21):5954–5958
Bian YS, Yan P, Osterheld MC, Fontolliet C, Benhattar J (2001) Promoter methylation analysis on microdissected paraffin-embedded tissues using bisulfite treatment and PCR-SSCP. Biotechniques 30(1):66–72
Warnecke PM, Stirzaker C, Song J, Grunau C, Melki JR, Clark SJ (2002) Identification and resolution of artifacts in bisulfite sequencing. Methods 27(2):101–107
Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG (1999) Inactivation of the DNA repair gene o6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 59(4):793–797
Harris LC, Potter PM, Tano K, Shiota S, Mitra S, Brent TP (1991) Characterization of the promoter region of the human o6-methylguanine-DNA methyltransferase gene. Nucleic Acids Res 19(22):6163–6167
Berginc G, Bracko M, Glavac D (2010) MS-MLPA reveals progressive age-dependent promoter methylation of tumor suppressor genes and possible role of IGSF4 gene in colorectal carcinogenesis of microsatellite instable tumors. Cancer Invest 28(1):94–102
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Glavač, D., Nardon, E. (2011). Qualitative Methylation Status Assessment. In: Stanta, G. (eds) Guidelines for Molecular Analysis in Archive Tissues. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-17890-0_30
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