Skip to main content
Top
Published in: BMC Cancer 1/2012

Open Access 01-12-2012 | Research article

A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples

Authors: Lampros Dimitrakopoulos, Panagiotis A Vorkas, Vasilis Georgoulias, Evi S Lianidou

Published in: BMC Cancer | Issue 1/2012

Login to get access

Abstract

Background

CST6 promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. The aim of our study was to develop a Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) assay for the investigation of CST6 promoter methylation.

Methods

We designed primers that amplify both methylated and unmethylated CST6 sequences after sodium bisulfate (SB) treatment and used spiked control samples of fully methylated to unmethylated SB converted genomic DNA to optimize the assay. We first evaluated the assay by analyzing 36 samples (pilot training group) and further analyzed 80 FFPES from operable breast cancer patients (independent group). MS-HRMA assay results for all 116 samples were compared with Methylation-Specific PCR (MSP) and the results were comparable.

Results

The developed assay is highly specific and sensitive since it can detect the presence of 1% methylated CST6 sequence and provides additionally a semi-quantitative estimation of CST6 promoter methylation. CST6 promoter was methylated in 39/80 (48.75%) of FFPEs with methylation levels being very different among samples. MS-HRMA and MSP gave comparable results when all samples were analyzed by both assays.

Conclusions

The developed MS-HRMA assay for CST6 promoter methylation is closed tube, highly sensitive, cost-effective, rapid and easy-to-perform. It gives comparable results to MSP in less time, while it offers the advantage of additionally providing an estimation of the level of methylation.
Appendix
Available only for authorised users
Literature
1.
go back to reference Laird PW: The power and promise of DNA methylation markers. Nat Rev Cancer. 2003, 3: 253-266. 10.1038/nrc1045.CrossRefPubMed Laird PW: The power and promise of DNA methylation markers. Nat Rev Cancer. 2003, 3: 253-266. 10.1038/nrc1045.CrossRefPubMed
2.
go back to reference Ferguson AT, Evron E, Umbricht CB, Pandita TK, Chan TA, Hermeking H, Marks JR, Lambers AR, Futreal PA, Stampfer MR, Sukumar S: High frequency of hypermethylation at the 14.3.3 sigma locus leads to gene silencing in breast cancer. Proc Natl Acad Sci USA. 2000, 97: 6049-6054. 10.1073/pnas.100566997.CrossRefPubMedPubMedCentral Ferguson AT, Evron E, Umbricht CB, Pandita TK, Chan TA, Hermeking H, Marks JR, Lambers AR, Futreal PA, Stampfer MR, Sukumar S: High frequency of hypermethylation at the 14.3.3 sigma locus leads to gene silencing in breast cancer. Proc Natl Acad Sci USA. 2000, 97: 6049-6054. 10.1073/pnas.100566997.CrossRefPubMedPubMedCentral
3.
go back to reference Esteller M: Epigenetics in cancer (Review). N Engl J Med. 2008, 358: 1148-1159. 10.1056/NEJMra072067.CrossRefPubMed Esteller M: Epigenetics in cancer (Review). N Engl J Med. 2008, 358: 1148-1159. 10.1056/NEJMra072067.CrossRefPubMed
4.
go back to reference Jones PA, Baylin SB: The fundamental role of epigenetic events in cancer. Nat Rev Genet. 2002, 3: 415-428.CrossRefPubMed Jones PA, Baylin SB: The fundamental role of epigenetic events in cancer. Nat Rev Genet. 2002, 3: 415-428.CrossRefPubMed
5.
go back to reference Esteller M: CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene. 2002, 21: 5427-5440. 10.1038/sj.onc.1205600.CrossRefPubMed Esteller M: CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene. 2002, 21: 5427-5440. 10.1038/sj.onc.1205600.CrossRefPubMed
6.
go back to reference Wojdacz TK, Dobrovic A, Hansen LL: Methylation-sensitive high-resolution melting. Nat Protoc. 2008, 3: 1903-1908. 10.1038/nprot.2008.191.CrossRefPubMed Wojdacz TK, Dobrovic A, Hansen LL: Methylation-sensitive high-resolution melting. Nat Protoc. 2008, 3: 1903-1908. 10.1038/nprot.2008.191.CrossRefPubMed
7.
go back to reference Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ: High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen. Clin Chem. 2003, 49: 853-860. 10.1373/49.6.853.CrossRefPubMed Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ: High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen. Clin Chem. 2003, 49: 853-860. 10.1373/49.6.853.CrossRefPubMed
8.
go back to reference Reed GH, Wittwer CT: Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem. 2004, 50: 1748-1754. 10.1373/clinchem.2003.029751.CrossRefPubMed Reed GH, Wittwer CT: Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem. 2004, 50: 1748-1754. 10.1373/clinchem.2003.029751.CrossRefPubMed
9.
go back to reference Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C: Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem. 2004, 50: 1156-1164. 10.1373/clinchem.2004.032136.CrossRefPubMed Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C: Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem. 2004, 50: 1156-1164. 10.1373/clinchem.2004.032136.CrossRefPubMed
10.
go back to reference Gundry CN, Dobrowolski SF, Martin YR, Robbins TC, Nay LM, Boyd N: Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons. Nucleic Acids Res. 2008, 36: 3401-3408. 10.1093/nar/gkn204.CrossRefPubMedPubMedCentral Gundry CN, Dobrowolski SF, Martin YR, Robbins TC, Nay LM, Boyd N: Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons. Nucleic Acids Res. 2008, 36: 3401-3408. 10.1093/nar/gkn204.CrossRefPubMedPubMedCentral
11.
go back to reference Krypuy M, Newnham GM, Thomas DM, Conron M, Dobrovic A: High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer. BMC Cancer. 2006, 6: 295-10.1186/1471-2407-6-295.CrossRefPubMedPubMedCentral Krypuy M, Newnham GM, Thomas DM, Conron M, Dobrovic A: High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer. BMC Cancer. 2006, 6: 295-10.1186/1471-2407-6-295.CrossRefPubMedPubMedCentral
12.
go back to reference Zhou L, Wang Y, Wittwer CT: Rare allele enrichment and detection by allele-specific PCR, competitive probe blocking and melting analysis. Biotechniques. 2011, 50: 311-318. Zhou L, Wang Y, Wittwer CT: Rare allele enrichment and detection by allele-specific PCR, competitive probe blocking and melting analysis. Biotechniques. 2011, 50: 311-318.
13.
go back to reference Wojdacz TK, Dobrovic A: Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res. 2007, 35: e41-10.1093/nar/gkm013.CrossRefPubMedPubMedCentral Wojdacz TK, Dobrovic A: Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res. 2007, 35: e41-10.1093/nar/gkm013.CrossRefPubMedPubMedCentral
14.
go back to reference Turk V, Bode W: The cystatins: protein inhibitors of cystein proteininases. FEBS Lett. 1991, 285: 213-219. 10.1016/0014-5793(91)80804-C.CrossRefPubMed Turk V, Bode W: The cystatins: protein inhibitors of cystein proteininases. FEBS Lett. 1991, 285: 213-219. 10.1016/0014-5793(91)80804-C.CrossRefPubMed
15.
go back to reference Sotiropoulou G, Anisowicz A, Sager R: Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer. J Biol Chem. 1997, 272: 903-910. 10.1074/jbc.272.2.903.CrossRefPubMed Sotiropoulou G, Anisowicz A, Sager R: Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer. J Biol Chem. 1997, 272: 903-910. 10.1074/jbc.272.2.903.CrossRefPubMed
16.
go back to reference Ni J, Abrahamson M, Zhang M, Fernandez MA, Grubb A, Su J, Yu GL, Li Y, Parmelee D, Xing L, Coleman TA, Gentz S, Thotakura R, Nguyen N, Hesselberg M, Gentz R: Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins. J Biol Chem. 1997, 272: 10853-10858. 10.1074/jbc.272.16.10853.CrossRefPubMed Ni J, Abrahamson M, Zhang M, Fernandez MA, Grubb A, Su J, Yu GL, Li Y, Parmelee D, Xing L, Coleman TA, Gentz S, Thotakura R, Nguyen N, Hesselberg M, Gentz R: Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins. J Biol Chem. 1997, 272: 10853-10858. 10.1074/jbc.272.16.10853.CrossRefPubMed
17.
go back to reference Stenman G, Astrom AK, Roijer E, Sotiropoulou G, Zhang M, Sager R: Assignment of a novel cysteine proteinase inhibitor (CST6) to 11q13 by fluorescence in situ hybridization. Cytogenet Cell Gent. 1997, 76: 45-46. 10.1159/000134512.CrossRef Stenman G, Astrom AK, Roijer E, Sotiropoulou G, Zhang M, Sager R: Assignment of a novel cysteine proteinase inhibitor (CST6) to 11q13 by fluorescence in situ hybridization. Cytogenet Cell Gent. 1997, 76: 45-46. 10.1159/000134512.CrossRef
18.
go back to reference Zeeuwen PL, Cheng T, Schalkwijk J: The biology of cystatin M/E and its cognate target proteases. J Invest Dermatol. 2009, 129: 1327-1338. 10.1038/jid.2009.40.CrossRefPubMed Zeeuwen PL, Cheng T, Schalkwijk J: The biology of cystatin M/E and its cognate target proteases. J Invest Dermatol. 2009, 129: 1327-1338. 10.1038/jid.2009.40.CrossRefPubMed
19.
go back to reference Cheng T, Hitomi K, van Vlijmen-Willems IM, de Jongh GJ, Yamamoto K, Nishi K, Watts C, Reinheckel T, Schalkwijk J, Zeeuwen PL: Cystatin M/E is a high affinity inhibitor of cathepsin V and cathepsin L by a reactive site that is distinct from the legumain-binding site. A novel clue for the role of cystatin M/E in epidermal cornification. J Biol Chem. 2006, 281: 15893-15899. 10.1074/jbc.M600694200.CrossRefPubMed Cheng T, Hitomi K, van Vlijmen-Willems IM, de Jongh GJ, Yamamoto K, Nishi K, Watts C, Reinheckel T, Schalkwijk J, Zeeuwen PL: Cystatin M/E is a high affinity inhibitor of cathepsin V and cathepsin L by a reactive site that is distinct from the legumain-binding site. A novel clue for the role of cystatin M/E in epidermal cornification. J Biol Chem. 2006, 281: 15893-15899. 10.1074/jbc.M600694200.CrossRefPubMed
20.
go back to reference Gawenda J, Traub F, Lück HJ, Kreipe H, von Wasielewski R: Legumain expression as a prognostic factor in breast cancer patients. Breast Cancer Res Treat. 2007, 102: 1-6. 10.1007/s10549-006-9311-z.CrossRefPubMed Gawenda J, Traub F, Lück HJ, Kreipe H, von Wasielewski R: Legumain expression as a prognostic factor in breast cancer patients. Breast Cancer Res Treat. 2007, 102: 1-6. 10.1007/s10549-006-9311-z.CrossRefPubMed
21.
go back to reference Murthy RV, Arbman G, Gao J, Roodman GD, Sun XF: Legumain expression in relation to clinicopathologic and biological variables in colorectal cancer. Clin Cancer Res. 2005, 11: 2293-2299. 10.1158/1078-0432.CCR-04-1642.CrossRefPubMed Murthy RV, Arbman G, Gao J, Roodman GD, Sun XF: Legumain expression in relation to clinicopathologic and biological variables in colorectal cancer. Clin Cancer Res. 2005, 11: 2293-2299. 10.1158/1078-0432.CCR-04-1642.CrossRefPubMed
22.
go back to reference Mohamed MM, Sloane BF: Cysteine cathepsins: multifunctional enzymes in cancer. Nat Rev Cancer. 2006, 6: 764-775. 10.1038/nrc1949.CrossRefPubMed Mohamed MM, Sloane BF: Cysteine cathepsins: multifunctional enzymes in cancer. Nat Rev Cancer. 2006, 6: 764-775. 10.1038/nrc1949.CrossRefPubMed
23.
go back to reference Ai L, Kim W, Kim TY, Fields CR, Massoll NA, Robertson KD, Brown KD: Epigenetic silencing of the tumor suppressor cystatin M occurs during breast cancer progression. Cancer Res. 2006, 66: 7899-7909. 10.1158/0008-5472.CAN-06-0576.CrossRefPubMed Ai L, Kim W, Kim TY, Fields CR, Massoll NA, Robertson KD, Brown KD: Epigenetic silencing of the tumor suppressor cystatin M occurs during breast cancer progression. Cancer Res. 2006, 66: 7899-7909. 10.1158/0008-5472.CAN-06-0576.CrossRefPubMed
24.
go back to reference Rivenbark AG, Jones WD, Coleman WB: DNA methylation-dependent silencing of CST6 in human breast cancer cell lines. Lab Invest. 2006, 86: 1233-1242. 10.1038/labinvest.3700485.CrossRefPubMed Rivenbark AG, Jones WD, Coleman WB: DNA methylation-dependent silencing of CST6 in human breast cancer cell lines. Lab Invest. 2006, 86: 1233-1242. 10.1038/labinvest.3700485.CrossRefPubMed
25.
go back to reference Schagdarsurengin U, Pfeifer GP, Dammann R: Frequent epigenetic inactivation of cystatin M in breast carcinoma. Oncogene. 2007, 26: 3089-3094. 10.1038/sj.onc.1210107.CrossRefPubMed Schagdarsurengin U, Pfeifer GP, Dammann R: Frequent epigenetic inactivation of cystatin M in breast carcinoma. Oncogene. 2007, 26: 3089-3094. 10.1038/sj.onc.1210107.CrossRefPubMed
26.
go back to reference Kioulafa M, Balkouranidou I, Sotiropoulou G, Kaklamanis L, Mavroudis D, Georgoulias V, Lianidou ES: Methylation of cystatin M promoter is associated with unfavorable prognosis in operable breast cancer. Int J Cancer. 2009, 125: 2887-2892. 10.1002/ijc.24686.CrossRefPubMed Kioulafa M, Balkouranidou I, Sotiropoulou G, Kaklamanis L, Mavroudis D, Georgoulias V, Lianidou ES: Methylation of cystatin M promoter is associated with unfavorable prognosis in operable breast cancer. Int J Cancer. 2009, 125: 2887-2892. 10.1002/ijc.24686.CrossRefPubMed
27.
go back to reference Chimonidou M, Strati A, Tzitzira A, Sotiropoulou G, Malamos N, Georgoulias V, Lianidou ES: DNA methylation of tumor suppressor and metastasis suppressor genes in circulating tumor cells. Clin Chem. 2011, 57: 1169-1177. 10.1373/clinchem.2011.165902.CrossRefPubMed Chimonidou M, Strati A, Tzitzira A, Sotiropoulou G, Malamos N, Georgoulias V, Lianidou ES: DNA methylation of tumor suppressor and metastasis suppressor genes in circulating tumor cells. Clin Chem. 2011, 57: 1169-1177. 10.1373/clinchem.2011.165902.CrossRefPubMed
28.
go back to reference Kroupis C, Christopoulos K, Devetzoglou M, Tsiagas I, Lianidou ES: Asymmetric real-time PCR detection of BRCA1 5382insC mutation by melting curve analysis in the LightCycler. Clin Chim Acta. 2008, 390: 141-144. 10.1016/j.cca.2007.12.024.CrossRefPubMed Kroupis C, Christopoulos K, Devetzoglou M, Tsiagas I, Lianidou ES: Asymmetric real-time PCR detection of BRCA1 5382insC mutation by melting curve analysis in the LightCycler. Clin Chim Acta. 2008, 390: 141-144. 10.1016/j.cca.2007.12.024.CrossRefPubMed
29.
go back to reference Warnecke PM, Stirzaker C, Melki JR, Millar DS, Paul CL, Clark SJ: Detection and measurement of PCR bias in quantitative methylation analysis of bisulphate-treated DNA. Nucleic Acids Res. 1997, 25: 4422-4426. 10.1093/nar/25.21.4422.CrossRefPubMedPubMedCentral Warnecke PM, Stirzaker C, Melki JR, Millar DS, Paul CL, Clark SJ: Detection and measurement of PCR bias in quantitative methylation analysis of bisulphate-treated DNA. Nucleic Acids Res. 1997, 25: 4422-4426. 10.1093/nar/25.21.4422.CrossRefPubMedPubMedCentral
30.
go back to reference Wojdacz TK, Borgbo T, Hansen LL: Primer design versus PCR bias in methylation independent PCR amplifications. Epigenetics. 2009, 4 (4): 231-234.CrossRefPubMed Wojdacz TK, Borgbo T, Hansen LL: Primer design versus PCR bias in methylation independent PCR amplifications. Epigenetics. 2009, 4 (4): 231-234.CrossRefPubMed
31.
go back to reference Wojdacz TK, Dobrovic A: Melting curve assays for DNA methylation analysis. Methods Mol Biol. 2009, 507: 229-240. 10.1007/978-1-59745-522-0_17.CrossRefPubMed Wojdacz TK, Dobrovic A: Melting curve assays for DNA methylation analysis. Methods Mol Biol. 2009, 507: 229-240. 10.1007/978-1-59745-522-0_17.CrossRefPubMed
32.
go back to reference Wojdacz TK, Hansen LL, Dobrovic A: A new approach to primer design for the control of PCR bias in methylation studies. BMC Res Notes. 2008, 1: 54-10.1186/1756-0500-1-54.CrossRefPubMedPubMedCentral Wojdacz TK, Hansen LL, Dobrovic A: A new approach to primer design for the control of PCR bias in methylation studies. BMC Res Notes. 2008, 1: 54-10.1186/1756-0500-1-54.CrossRefPubMedPubMedCentral
33.
go back to reference Wojdacz TK, Hansen LL: Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay. Biotechniques. 2006, 41: 274, 276-278.CrossRef Wojdacz TK, Hansen LL: Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay. Biotechniques. 2006, 41: 274, 276-278.CrossRef
34.
go back to reference Rivenbark AG, Livasy CA, Boyd CE, Keppler D, Coleman WB: Methylation-dependent silencing of CST6 in primary human breast tumors and metastatic lesions. Exp Mol Pathol. 2007, 83: 188-197. 10.1016/j.yexmp.2007.03.008.CrossRefPubMedPubMedCentral Rivenbark AG, Livasy CA, Boyd CE, Keppler D, Coleman WB: Methylation-dependent silencing of CST6 in primary human breast tumors and metastatic lesions. Exp Mol Pathol. 2007, 83: 188-197. 10.1016/j.yexmp.2007.03.008.CrossRefPubMedPubMedCentral
35.
go back to reference Kundel HL, Polansky M: Measurement of observer agreement. Radiology. 2003, 228: 303-308. 10.1148/radiol.2282011860.CrossRefPubMed Kundel HL, Polansky M: Measurement of observer agreement. Radiology. 2003, 228: 303-308. 10.1148/radiol.2282011860.CrossRefPubMed
36.
go back to reference Qiu J, Ai L, Ramachandran C, Yao B, Gopalakrishnan S, Fields CR, Delmas AL, Dyer LM, Melnick SJ, Yachnis AT, Schwartz PH, Fine HA, Brown KD, Robertson KD: Invasion suppressor cystatin E/M (CST6): high-level cell type-specific expression in normal brain and epigenetic silencing in gliomas. Lab Invest. 2008, 88: 910-925. 10.1038/labinvest.2008.66.CrossRefPubMedPubMedCentral Qiu J, Ai L, Ramachandran C, Yao B, Gopalakrishnan S, Fields CR, Delmas AL, Dyer LM, Melnick SJ, Yachnis AT, Schwartz PH, Fine HA, Brown KD, Robertson KD: Invasion suppressor cystatin E/M (CST6): high-level cell type-specific expression in normal brain and epigenetic silencing in gliomas. Lab Invest. 2008, 88: 910-925. 10.1038/labinvest.2008.66.CrossRefPubMedPubMedCentral
37.
go back to reference Veena MS, Lee G, Keppler D, Mendonca MS, Redpath JL, Stanbridge EJ, Wilczynski SP, Srivatsan ES: Inactivation of the cystatin E/M tumor suppressor gene in cervical cancer. Genes Chromosomes Cancer. 2008, 47: 740-754. 10.1002/gcc.20576.CrossRefPubMedPubMedCentral Veena MS, Lee G, Keppler D, Mendonca MS, Redpath JL, Stanbridge EJ, Wilczynski SP, Srivatsan ES: Inactivation of the cystatin E/M tumor suppressor gene in cervical cancer. Genes Chromosomes Cancer. 2008, 47: 740-754. 10.1002/gcc.20576.CrossRefPubMedPubMedCentral
38.
go back to reference Pulukuri SM, Gorantla B, Knost JA, Rao JS: Frequent loss of cystatin E/M expression implicated in the progression of prostate cancer. Oncogene. 2009, 28: 2829-2838. 10.1038/onc.2009.134.CrossRefPubMedPubMedCentral Pulukuri SM, Gorantla B, Knost JA, Rao JS: Frequent loss of cystatin E/M expression implicated in the progression of prostate cancer. Oncogene. 2009, 28: 2829-2838. 10.1038/onc.2009.134.CrossRefPubMedPubMedCentral
39.
go back to reference Lin HJ, et al: Breast cancer-associated fibroblasts confer AKT1-mediated epigenetic silencing of Cystatin M in epithelial cells. Cancer Res. 2008, 68: 10257-10266. 10.1158/0008-5472.CAN-08-0288.CrossRefPubMedPubMedCentral Lin HJ, et al: Breast cancer-associated fibroblasts confer AKT1-mediated epigenetic silencing of Cystatin M in epithelial cells. Cancer Res. 2008, 68: 10257-10266. 10.1158/0008-5472.CAN-08-0288.CrossRefPubMedPubMedCentral
40.
go back to reference Radpour R, Kohler C, Haghighi MM, Fan AX, Holzgreve W, Zhong XY: Methylation profiles of 22 candidate genes in breast cancer using high-throughput MALDI-TOF mass array. Oncogene. 2009, 28: 2969-2978. 10.1038/onc.2009.149.CrossRefPubMed Radpour R, Kohler C, Haghighi MM, Fan AX, Holzgreve W, Zhong XY: Methylation profiles of 22 candidate genes in breast cancer using high-throughput MALDI-TOF mass array. Oncogene. 2009, 28: 2969-2978. 10.1038/onc.2009.149.CrossRefPubMed
41.
go back to reference Rodenhiser DI, Andrews J, Kennette W, Sadikovic B, Mendlowitz A, Tuck AB, Chambers AF: Epigenetic mapping and functional analysis in a breast cancer metastasis model using whole-genome promoter tiling microarrays. Breast Cancer Res. 2008, 10: R62-10.1186/bcr2121.CrossRefPubMedPubMedCentral Rodenhiser DI, Andrews J, Kennette W, Sadikovic B, Mendlowitz A, Tuck AB, Chambers AF: Epigenetic mapping and functional analysis in a breast cancer metastasis model using whole-genome promoter tiling microarrays. Breast Cancer Res. 2008, 10: R62-10.1186/bcr2121.CrossRefPubMedPubMedCentral
42.
go back to reference Morris MR, Ricketts C, Gentle D, Abdulrahman M, Clarke N, Brown M, Kishida T, Yao M, Latif F, Maher ER: Identification of candidate tumour suppressor genes frequently methylated in renal cell carcinoma. Oncogene. 2010, 29: 2104-2117. 10.1038/onc.2009.493.CrossRefPubMedPubMedCentral Morris MR, Ricketts C, Gentle D, Abdulrahman M, Clarke N, Brown M, Kishida T, Yao M, Latif F, Maher ER: Identification of candidate tumour suppressor genes frequently methylated in renal cell carcinoma. Oncogene. 2010, 29: 2104-2117. 10.1038/onc.2009.493.CrossRefPubMedPubMedCentral
43.
go back to reference Ko E, Park SE, Cho EY, Kim Y, Hwang JA, Lee YS, Nam SJ, Bang S, Park J, Kim DH: Cystatin M loss is associated with the losses of estrogen receptor, progesterone receptor, and HER4 in invasive breast cancer. Breast Cancer Res. 2010, 12: R100-10.1186/bcr2783.CrossRefPubMedPubMedCentral Ko E, Park SE, Cho EY, Kim Y, Hwang JA, Lee YS, Nam SJ, Bang S, Park J, Kim DH: Cystatin M loss is associated with the losses of estrogen receptor, progesterone receptor, and HER4 in invasive breast cancer. Breast Cancer Res. 2010, 12: R100-10.1186/bcr2783.CrossRefPubMedPubMedCentral
44.
go back to reference Dahl C, Guldberg P: High-Resolution Melting for Accurate Assessment of DNA Methylation. Clin Chem (editorial). 2007, 53: 1877-1878. 10.1373/clinchem.2007.094854.CrossRef Dahl C, Guldberg P: High-Resolution Melting for Accurate Assessment of DNA Methylation. Clin Chem (editorial). 2007, 53: 1877-1878. 10.1373/clinchem.2007.094854.CrossRef
45.
go back to reference Worm J, Aggerholm A, Guldberg P: In-tube DNA methylation profiling by fluorescence melting curve analysis. Clin Chem. 2001, 47: 1183-1189.PubMed Worm J, Aggerholm A, Guldberg P: In-tube DNA methylation profiling by fluorescence melting curve analysis. Clin Chem. 2001, 47: 1183-1189.PubMed
46.
go back to reference Guldberg P, Worm J, Grønbæk K: Profiling DNA methylation by melting analysis. Methods. 2002, 27: 121-127. 10.1016/S1046-2023(02)00063-4.CrossRefPubMed Guldberg P, Worm J, Grønbæk K: Profiling DNA methylation by melting analysis. Methods. 2002, 27: 121-127. 10.1016/S1046-2023(02)00063-4.CrossRefPubMed
47.
go back to reference Dahl C, Grønskov K, Larsen LA, Guldberg P, Brøndum-Nielsen K: A homogeneous assay for analysis of FMR1 promoter methylation in patients with fragile X syndrome. Clin Chem. 2007, 53: 790-793. 10.1373/clinchem.2006.080762.CrossRefPubMed Dahl C, Grønskov K, Larsen LA, Guldberg P, Brøndum-Nielsen K: A homogeneous assay for analysis of FMR1 promoter methylation in patients with fragile X syndrome. Clin Chem. 2007, 53: 790-793. 10.1373/clinchem.2006.080762.CrossRefPubMed
Metadata
Title
A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples
Authors
Lampros Dimitrakopoulos
Panagiotis A Vorkas
Vasilis Georgoulias
Evi S Lianidou
Publication date
01-12-2012
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2012
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/1471-2407-12-486

Other articles of this Issue 1/2012

BMC Cancer 1/2012 Go to the issue
Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras
Prof. Fabrice Barlesi
Developed by: Springer Medicine