Abstract 808: Quantitative digital PCR analysis of cancer gene promoter methylation using low amounts of input DNA.
Authors: Samuels Michael L. , Kemp Ryan , Long Frances , Petrisko Jill , Nguyen Lam , Krispin Manuel 
Published: August 2014

Silencing of tumor suppressor and tumor-related gene transcription by hypermethylation at promoter CpG motifs is a significant mechanism at work in human tumorgenesis. Various methods have been developed to analyze DNA methylation levels, both across the genome and at specific loci, in order to discover and interrogate disease relevant loci for methylation-based transcriptional control. Methods able to quantitatively measure differences in DNA methylation between normal and cancer cells provide promising sources for biomarker identification and assessment.

Here we report on the use of a methylation assay (Zymo Research's OneStep qMethyl™) and digital PCR counting (RainDrop™ dPCR) to enable quantitative measurements of DNA methylation at specific genomic loci using small amounts of non-bisulfite converted DNA. MGMT promoter methylation measurements are used to illustrate the method's robust quantification of small percent methylation changes using low amounts of input DNA.

Zymo Research's OneStep qMethyl™ Kit is used for the detection of region-specific DNA methylation via the selective amplification of methylated cytosines after digestion with methylation-sensitive restriction enzymes. RainDance Technologies’ RainDrop™ dPCR system is a highly sensitive tool for precise quantification of nucleic acids using probe-based qPCR reagents. RainDrop offers unique analytical advantages for clinical diagnostics due to its exceptionally high sensitivity for absolute quantification and capability to multiplex assays, using a wide dynamic range of input DNA in a contamination-free and simple workflow. The combination of both technologies enables a novel, fast, and robust method for quantitative methylation analysis of small amounts of DNA.

The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) removes mutagenic alkyl adducts from DNA, and its epigenetic silencing by promoter methylation confers susceptibility to DNA-damaging alkylating agents in glioblastomas, melanomas, and lung cancer cell lines, and is increased in cancers including advanced gastric carcinomas, colorectal tumors, and triple-negative breast cancers. A sensitive, quick, and cost-effective quantitative method for assessing MGMT promoter methylation would greatly enhance the diagnostic potential for this candidate cancer biomarker.

Here we show digital quantification of MGMT promoter methylation across the range from 0 to 100%, demonstrating a linear correlation that enables less than 15% methylation changes to be robustly measured using less than 20 nanograms of input DNA. This method provides a sensitive count of region-specific methylation percentages using an easy workflow. Continuing work will make use of RainDrop's unique ability to multiplex assays to enable combining MGMT methylation analysis with additional cancer relevant methylation biomarkers in a single assay.

Citation Format: Michael L. Samuels, Ryan Kemp, Frances Long, Jill Petrisko, Lam Nguyen, Manuel Krispin. Quantitative digital PCR analysis of cancer gene promoter methylation using low amounts of input DNA. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 808. doi:10.1158/1538-7445.AM2013-808