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


CONCLUSIONS
We propose MCA-MSP and MCA-Meth as alternative or supplementary techniques to MSP or bisulfite sequencing.
Lorente
Aiala
A
Brain Tumor Biology Unit, University of Navarra, Pamplona, Spain. altrigos@alumni.unav.es
Mueller
Wolf
W
Urdangarín
Edurne
E
Lázcoz
Paula
P
von Deimling
Andreas
A
Castresana
Javier S
JS
D045744:Cell Line, Tumor; D003598:Cytoskeletal Proteins; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics; D045643:DNA Repair Enzymes / Q000235:genetics; D005240:Feasibility Studies; D015870:Gene Expression; D016147:Genes, Tumor Suppressor; D006801:Humans; D017599:Neuroectodermal Tumors / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000235:genetics
D003598:Cytoskeletal Proteins; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; C472720:ZMYND10 protein, human; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes