MethyLight

Abstract

MethyLight is a sodium-bisulfite-dependent, quantitative, fluorescence-based, real-time PCR method to sensitively detect and quantify DNA methylation in genomic DNA. MethyLight relies on methylation-specific priming combined with methylation-specific fluorescent probing. This combination of methylation-specific detection principles results in a highly methylation-specific detection technology, with an accompanying ability to sensitively detect very low frequencies of hypermethylated alleles. The high sensitivity and specificity of MethyLight make it uniquely well suited for detection of low-frequency DNA methylation biomarkers as evidence of disease. At the same time, the quantitative accuracy of real-time PCR and the flexibility to design bisulfite-dependent, methylation-independent control reactions allows for a quantitative assessment of these low-frequency methylation events. We describe the experimental steps of MethyLight analysis in detail. Furthermore, we present here principles and design examples for three types of quality-control reactions. QC-1 reactions are methylation-independent reactions to monitor sample quantity and integrity. QC-2 reactions are bisulfite-independent reactions to monitor recovery efficiencies of the bisulfite-conversion methodology used. QC-3 reactions are bisulfite-independent primed reactions with variable bisulfite-dependent probing to monitor completeness of the sodium bisulfite treatment. We show that these control reactions perform as expected in a time-course experiment interrupting sodium bisulfite conversion at various timepoints.

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

“…Quantification of total bisulfite DNA copies for the standard and in each sample was determined by reference to the C-Less assay as described previously. 37,38 Purified CD3 T cells were obtained from healthy blood donors using antibody based negative selection and magnetic bead technology (Miltenyi, San Diego, CA). 5 Purity of the T cells was confirmed by FACS analysis.…”

“…We control for the total input of methylated and demethylated DNA by using a gene reference target that is insensitive to CpG methylation status (referred to as the C-less reaction). 37,38 An important application of this approach is in oncology patients who demonstrate T cell defects. It has also been recognized for many years that glioma patients, and particularly Stage IV glioblastoma (GBM) patients, show peripheral immune defects affecting T cells.…”

Distinct DNA methylation alterations are associated with cribriform architecture and intraductal carcinoma in Gleason pattern 4 prostate tumors

“…CpGenome is a commercially available fully methylated DNA (EMD Millipore, Billerica, MA, USA). ALU was a control reaction that measured the level of input DNA and normalized the signal for each methylation reaction (27). categories.…”

198 Hsa-mir-124-3 CpG island methylation is associated with advanced tumours and disease recurrence of patients with clear cell renal cell carcinoma

“…Bisulphite conversion of DNA was performed as described before (Peters et al, 2007). To control bisulphite conversion efficiency, all samples were subjected to quantitative methylation-specific PCRs (qMSP) using the concept reported by Campan et al (2009) andvan der Horst et al (2004). Briefly, detection of the methylation-independent repetitive sequence ALU-C4 (QC1) and a single copy b-Actin (ACTB) (QC2) sequence served to measure the yield of conversion and to provide for controlled input of bisulphite converted template DNA into qMSP analyses, while the repetitive sequence ALU-YB8 (QC3) allows to quantify unconverted DNA.…”