A methylation sensitive dot blot assay (MS-DBA) for the quantitative analysis of DNA methylation in clinical samples

Abstract

Background: There is increasing interest in DNA methylation and in its implication in transcriptional gene silencing, a phenomenon commonly seen in human cancer.

Aims: To develop a new method that would allow quantitative DNA methylation analysis in a large range of clinical samples, independently of the processing protocol.

Methods: A methylation sensitive dot blot assay (MS-DBA) was developed, which is quantitative and combines bisulfite modification, PCR amplification using primers without CpG sites, and dot blot analysis with two probes specific for methylated and unmethylated DNA.

Results: The established method was used to study methylation of the hTERT, APC, and p16 promoter regions in microdissected, formalin fixed and paraffin wax embedded tissues.

Conclusions: MS-DBA is a sensitive, specific, and quantitative approach to analyse DNA methylation in a variety of frozen or fixed tissues. Moreover, MS-DBA is rapid, easy to perform, and permits the screening of a large panel of samples in one experiment. Thus, MS-DBA can facilitate the routine analysis of DNA methylation in all types of clinical samples.

Figure 1

Outline of the methylation sensitive dot blot assay. The method is based on bisulfite modification, polymerase chain reaction amplification using primers without CpG sites and specific to the modified strand, followed by a quantitative, non-radioactive dot blot readout. The use of two DIG labelled oligoprobes specific for either the methylated or the unmethylated DNA leads to different readout patterns: no methylation, full methylation, or a combination of unmethylated and methylated alleles.

Figure 2

Determination of quantitative methylation by methylation sensitive dot blot assay (MS-DBA) for the hTERT promoter region. DNA from normal placental tissue was methylated with SssI methylase. SssI methylated and unmethylated DNAs were mixed at different ratios as indicated above the dot blot. The data show that MS-DBA yields reliable results across a wide range of DNA methylation.

Figure 3

MS-DBA of hTERT, APC, and p16 promoter regions in human oesophageal tissues. N1 and N2, normal squamous tissues; T1–10, oesophageal adenocarcinomas. An external standard with 0%, 50%, and 100% methylation was used as control.