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BMC Cancer
Published in: BMC Cancer 1/2008

Open Access 01-12-2008 | Technical advance

Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR

Authors: Aiala Lorente, Wolf Mueller, Edurne Urdangarín, Paula Lázcoz, Andreas von Deimling, Javier S Castresana

Published in: BMC Cancer | Issue 1/2008

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Abstract

Background

We present two melting curve analysis (MCA)-based semiquantitative real time PCR techniques to detect the promoter methylation status of genes. The first, MCA-MSP, follows the same principle as standard MSP but it is performed in a real time thermalcycler with results being visualized in a melting curve. The second, MCA-Meth, uses a single pair of primers designed with no CpGs in its sequence. These primers amplify both unmethylated and methylated sequences. In clinical applications the MSP technique has revolutionized methylation detection by simplifying the analysis to a PCR-based protocol. MCA-analysis based techniques may be able to further improve and simplify methylation analyses by reducing starting DNA amounts, by introducing an all-in-one tube reaction and by eliminating a final gel stage for visualization of the result. The current study aimed at investigating the feasibility of both MCA-MSP and MCA-Meth in the analysis of promoter methylation, and at defining potential advantages and shortcomings in comparison to currently implemented techniques, i.e. bisulfite sequencing and standard MSP.

Methods

The promoters of the RASSF1A (3p21.3), BLU (3p21.3) and MGMT (10q26) genes were analyzed by MCA-MSP and MCA-Meth in 13 astrocytoma samples, 6 high grade glioma cell lines and 4 neuroblastoma cell lines. The data were compared with standard MSP and validated by bisulfite sequencing.

Results

Both, MCA-MSP and MCA-Meth, successfully determined promoter methylation. MCA-MSP provided information similar to standard MSP analyses. However the analysis was possible in a single tube and avoided the gel stage. MCA-Meth proved to be useful in samples with intermediate methylation status, reflected by a melting curve position shift in dependence on methylation extent.

Conclusion

We propose MCA-MSP and MCA-Meth as alternative or supplementary techniques to MSP or bisulfite sequencing.
Appendix
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Metadata
Title
Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR
Authors
Aiala Lorente
Wolf Mueller
Edurne Urdangarín
Paula Lázcoz
Andreas von Deimling
Javier S Castresana
Publication date
01-12-2008
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2008
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/1471-2407-8-61

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