Comparison between Absolute and Relative Quantification of LINE-1 Methylation Level in Breast Cancer
Main Article Content
Abstract
There are two accurate formulas corresponding to absolute and relative methods that were widely used for quantitative DNA methylation level. In the absolute method, DNA methylation is examined by two sets of MSP primers, one is specific for the methylated state and the other for the unmethylated state of the target. In the relative method, DNA methylation is examined by two sets of primers, one is MSP, specific for the methylated state, and the other is MIP, for the measurement of input DNA levels to normalize the signal for each methylation reaction. Both these methods, subjected to quantitative measurement of the LINE-1 methylation level in breast cancer samples, indicating LINE-1 hypermethylation in tumour tissue samples as compared with adjacent tissue samples. The methylated LINE-1 amount is enormous but the unmethylated one is scant, consequently leading to greater measurement variance compared with the comparative quantification.Thus, the relative quantification, may be a more convenient method for quantitatively measuring the LINE-1 methylation level.
Keywords:
DNA methylation, absolute quantification qPCR, relative quantification qPCR.
References
[1] T. D. Schmittgen, K. J. Livak, Analyzing Real-time PCR Data by the Comparative C(T) Method, Nature Protocols, Vol. 3, 2008, pp. 1101-1108.
[2] J. Ibrahim, M. Peeters, G. Van Camp, K. Op de Beeck, Methylation Biomarkers for Early Cancer Detection and Diagnosis: Current and Future Perspectives, European Journal of Cancer, Vol. 178, 2023, pp. 91-113.
[3] L. S. Kristensen, L. L. Hansen, PCR-based Methods for Detecting Single-locus DNA Methylation Biomarkers in Cancer Diagnostics, Prognostics, and Response to Treatment, Clinical Chemistry, Vol. 55, 2009, pp. 1471-1483.
[4] S. J. Clark, J. Harrison, C. L. Paul, M. Frommer, High Sensitivity Mapping of Methylated Cytosines, Nucleic Acids Research, Vol. 22, 1994, pp. 2990-2997.
[5] J. G. Herman, J. R. Graff, S. Myöhänen, B. D. Nelkin, S. B. Baylin, Methylation-specific PCR: A Novel PCR Assay for Methylation Status of CpG Islands, Proceedings of the National Academy Sciences of the USA, Vol. 93, 1996,
pp. 9821-9826.
[6] H. Gudnason H, D. Martin, B. B. Bang, A. Wolff, Comparison of Multiple DNA Dyes for Real-time PCR: Effects of Dye Concentration and Sequence Composition on DNA Amplification and Melting Temperature, Nucleic Acids Research, Vol. 35, 2007, pp. e127.
[7] S. Giglio, P. Monis, C. Saint, Demonstration of Preferential Binding of SYBR Green I to Specific DNA Fragments in Real-time Multiplex PCR, Nucleic Acids Res, Vol. 31, 2003, pp. e136.
[8] K. J. Livak, T. D. Schmittgen, Analysis of Relative Gene Expression Data using Real-time Quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods. Vol. 25, 2001, pp. 402-408.
[9] H. Thomassin, C. Kress, T. Grange, MethylQuant: A Sensitive Method for Quantifying Methylation of Specific Cytosines within the Genome, Nucleic Acids Research, Vol. 32, 2004, pp. e168.
[10] E. S. Lander et al., International Human Genome Sequencing Consortium, Initial Sequencing and Analysis of the Human Genome, Nature, Vol. 409, 2001, pp. 860-921.
[11] D. A. T. Pham, S. D. Le, T. M. Doan, P. T. Luu, U. Q. Nguyen, S. V. Ho, L. T. T. Vo, Standardization of DNA Amount for Bisulfite Conversion for Analyzing the Methylation Status of LINE-1 in Lung Cancer, PloS one, Vol. 16, 2021, pp. e0256254.
[12] T. T. Q. Trang, P. T. Tung, N. T. Than, D. H Trang, L. T. T. Phuong, N. Q. Uyen, V. D. Linh, N. N. Quang, H. V. Son, D. V. Hang, H. V. Tong, V. T. T Lan, An Appropriate DNA Input for Bisulfite Conversion Reveals LINE-1 and Alu Hypermethylation in Tissues and Circulating Cell-free DNA from Cancers, PLoS One, Vol. 19, 2924, pp. e0316394.
[13] C. A. Eads, K. D. Danenberg, K. Kawakami, L. B. Saltz, C. Blake, D. Shibata, P. V. Danenberg, P. W. Laird, MethyLight: A High-throughput Assay to Measure DNA Methylation, Nucleic Acids Research, Vol. 28, 2000, pp. e32.
[14] S. Jin et al., Efficient Detection and Post-surgical Monitoring of Colon Cancer with a Multi-marker DNA Methylation Liquid Biopsy, Proceedings of the National Academy Sciences of the USA. Vol. 118, 2021, pp. e20174-21118.
[15] D. J. Weisenberger, M. Campan, T. I. Long, M. Kim, C. Woods, E. Fiala, M. Ehrlich, P. W. Laird, Analysis of Repetitive Element DNA Methylation by MethyLight, Nucleic Acids Research, Vol. 33, 2005, pp. 6823-6836.
[16] T. K. Wojdacz, L. L. Hansen, Reversal of PCR Bias for Improved Sensitivity of the DNA Methylation Melting Curve Assay, BioTechniques, Vol. 41, 2006, pp. 274-278.
[17] L. Ji, T. Sasaki, X. Sun, P. Ma, Z. A. Lewis, R. J. Schmitz, Methylated DNA is Over-represented in Whole-genome Bisulfite Sequencing Data, Frontiers in Genetics, Vol. 5, 2014, pp. 341.
[18] Š. Šestáková, C. Šálek, H. Remešová, DNA Methylation Validation Methods: A Coherent Review with Practical Comparison, Biological Procedures Online, Vol. 21, 2019, pp. 19.
[19] M. Barchitta, A. Quattrocchi, A. Maugeri, LINE-1 Hypermethylation in White Blood Cell DNA is Associated with High-grade Cervical Intraepithelial Neoplasia, BMC Cancer, Vol. 17, 2017, pp. 601.
[20] K. Y. Neven, M. Piola, L. Angelici, Repetitive Element Hypermethylation in Multiple Sclerosis Patients, BMC Genet, Vol. 17, 2016, pp. 84.
[21] M. M. Kelsey, Reconsidering LINE-1’s Role in Cancer: Does LINE-1 Function as a Reporter Detecting Early Cancer-associated Epigenetic Signatures? Evolution, Medicine, and Public Health, Vol. 9, 2021, pp. 78.
[2] J. Ibrahim, M. Peeters, G. Van Camp, K. Op de Beeck, Methylation Biomarkers for Early Cancer Detection and Diagnosis: Current and Future Perspectives, European Journal of Cancer, Vol. 178, 2023, pp. 91-113.
[3] L. S. Kristensen, L. L. Hansen, PCR-based Methods for Detecting Single-locus DNA Methylation Biomarkers in Cancer Diagnostics, Prognostics, and Response to Treatment, Clinical Chemistry, Vol. 55, 2009, pp. 1471-1483.
[4] S. J. Clark, J. Harrison, C. L. Paul, M. Frommer, High Sensitivity Mapping of Methylated Cytosines, Nucleic Acids Research, Vol. 22, 1994, pp. 2990-2997.
[5] J. G. Herman, J. R. Graff, S. Myöhänen, B. D. Nelkin, S. B. Baylin, Methylation-specific PCR: A Novel PCR Assay for Methylation Status of CpG Islands, Proceedings of the National Academy Sciences of the USA, Vol. 93, 1996,
pp. 9821-9826.
[6] H. Gudnason H, D. Martin, B. B. Bang, A. Wolff, Comparison of Multiple DNA Dyes for Real-time PCR: Effects of Dye Concentration and Sequence Composition on DNA Amplification and Melting Temperature, Nucleic Acids Research, Vol. 35, 2007, pp. e127.
[7] S. Giglio, P. Monis, C. Saint, Demonstration of Preferential Binding of SYBR Green I to Specific DNA Fragments in Real-time Multiplex PCR, Nucleic Acids Res, Vol. 31, 2003, pp. e136.
[8] K. J. Livak, T. D. Schmittgen, Analysis of Relative Gene Expression Data using Real-time Quantitative PCR and the 2(-Delta Delta C(T)) Method, Methods. Vol. 25, 2001, pp. 402-408.
[9] H. Thomassin, C. Kress, T. Grange, MethylQuant: A Sensitive Method for Quantifying Methylation of Specific Cytosines within the Genome, Nucleic Acids Research, Vol. 32, 2004, pp. e168.
[10] E. S. Lander et al., International Human Genome Sequencing Consortium, Initial Sequencing and Analysis of the Human Genome, Nature, Vol. 409, 2001, pp. 860-921.
[11] D. A. T. Pham, S. D. Le, T. M. Doan, P. T. Luu, U. Q. Nguyen, S. V. Ho, L. T. T. Vo, Standardization of DNA Amount for Bisulfite Conversion for Analyzing the Methylation Status of LINE-1 in Lung Cancer, PloS one, Vol. 16, 2021, pp. e0256254.
[12] T. T. Q. Trang, P. T. Tung, N. T. Than, D. H Trang, L. T. T. Phuong, N. Q. Uyen, V. D. Linh, N. N. Quang, H. V. Son, D. V. Hang, H. V. Tong, V. T. T Lan, An Appropriate DNA Input for Bisulfite Conversion Reveals LINE-1 and Alu Hypermethylation in Tissues and Circulating Cell-free DNA from Cancers, PLoS One, Vol. 19, 2924, pp. e0316394.
[13] C. A. Eads, K. D. Danenberg, K. Kawakami, L. B. Saltz, C. Blake, D. Shibata, P. V. Danenberg, P. W. Laird, MethyLight: A High-throughput Assay to Measure DNA Methylation, Nucleic Acids Research, Vol. 28, 2000, pp. e32.
[14] S. Jin et al., Efficient Detection and Post-surgical Monitoring of Colon Cancer with a Multi-marker DNA Methylation Liquid Biopsy, Proceedings of the National Academy Sciences of the USA. Vol. 118, 2021, pp. e20174-21118.
[15] D. J. Weisenberger, M. Campan, T. I. Long, M. Kim, C. Woods, E. Fiala, M. Ehrlich, P. W. Laird, Analysis of Repetitive Element DNA Methylation by MethyLight, Nucleic Acids Research, Vol. 33, 2005, pp. 6823-6836.
[16] T. K. Wojdacz, L. L. Hansen, Reversal of PCR Bias for Improved Sensitivity of the DNA Methylation Melting Curve Assay, BioTechniques, Vol. 41, 2006, pp. 274-278.
[17] L. Ji, T. Sasaki, X. Sun, P. Ma, Z. A. Lewis, R. J. Schmitz, Methylated DNA is Over-represented in Whole-genome Bisulfite Sequencing Data, Frontiers in Genetics, Vol. 5, 2014, pp. 341.
[18] Š. Šestáková, C. Šálek, H. Remešová, DNA Methylation Validation Methods: A Coherent Review with Practical Comparison, Biological Procedures Online, Vol. 21, 2019, pp. 19.
[19] M. Barchitta, A. Quattrocchi, A. Maugeri, LINE-1 Hypermethylation in White Blood Cell DNA is Associated with High-grade Cervical Intraepithelial Neoplasia, BMC Cancer, Vol. 17, 2017, pp. 601.
[20] K. Y. Neven, M. Piola, L. Angelici, Repetitive Element Hypermethylation in Multiple Sclerosis Patients, BMC Genet, Vol. 17, 2016, pp. 84.
[21] M. M. Kelsey, Reconsidering LINE-1’s Role in Cancer: Does LINE-1 Function as a Reporter Detecting Early Cancer-associated Epigenetic Signatures? Evolution, Medicine, and Public Health, Vol. 9, 2021, pp. 78.