APPLICATION NOTE
EpiMelt Real-Time PCR Master Mix

Abstract

Epigenetic biomarkers are becoming increasingly relevant in the investigation of disease predisposition, screening and diagnosis, treatment guidance, and post-treatment surveillance. As the presence or absence of these epigenetic biomarkers greatly impacts the disease course and management, the sensitivity of the methylation detection methods is crucial.

The EpiMelt Methylation Detection Assays are based on the Methylation-Sensitive High-Resolution Melting (MSHRM) technology. The unique primer design and specific annealing temperature ensure a robust and highly sensitive DNA methylation analysis. In this application note, we show the performance of three EpiMelt Methylation Detection Assays using the EpiMelt Real-Time PCR Master Mix and demonstrate this combination to be a precise and sensitive method of methylation detection.

Introduction

DNA methylation is an epigenetic alteration of DNA, which plays an important role in the regulation of many biological processes, including gene expression and silencing, genomic imprinting, X chromosome inactivation, and genome stability1,2. An aberrant methylation pattern is a hallmark of cancer. Alteration of the established methylation pattern is typically an early event in carcinogenesis, leading to epigenetic silencing of tumor suppressor genes and transcriptional activation of oncogenes1. The methylation status at specific genes can have distinct consequences for the disease course, and epigenetic biomarkers have potential in all stages of disease. Before diagnosis, people can be predisposed to certain cancers through germline genetic and epigenetic alterations, and detection of DNA methylation at specific loci is one way to determine cancer predisposition.

Multiple studies have demonstrated BRCA1 hypermethylation in peripheral blood predisposing to ovarian and breast cancer3-5. Epigenetic biomarkers have also been used for screening and diagnostic purposes.

In the age of personalized medicine, methylation biomarkers can also help guide treatment choices, thereby optimizing the effect for some patients, while sparing other patients from unnecessary side effects. In breast and ovarian cancer, the methylation status of BRCA1 can guide the choice of PARP inhibitor treatment7,8. Finally, the detection of methylation biomarkers is a sensitive mode of post-treatment surveillance. In bladder cancer, monitoring methylation markers in urine improves the detection of recurrent disease and may spare bladder cancer patients from disease monitoring through more invasive, less sensitive methods9.

The EpiMelt Methylation Detection Assays are based on the technology of Methylation-Sensitive High-Resolution Melting (MS-HRM). In MS-HRM, sequence-specific melting profiles of amplicons are observed across a gradually increasing temperature gradient in the presence of an intercalating, fluorescent dye10,11. The amplicons are amplified from bisulfite-converted template DNA, so amplicons from methylated template DNA will have a high GC content, and amplicons from the unmethylated template will have a low GC content. The amplification is performed using primers that match the methylated template and have a small mismatch towards the unmethylated template. This unique primer design is employed to overcome PCR bias12. Amplicons from the methylated template will then melt at a higher temperature than amplicons from the unmethylated template10. This principle allows the identification of methylation status based on the amplicon melting profile.

The EpiMelt Methylation Detection Assays include a locus-specific primer mix, and three ready-to-use controls: A Methylation Positive Control, an Assay Calibration Control, and a Methylation Negative Control. The method is semiquantitative, and the DNA methylation status of an unknown sample is interrogated by comparing the melting profile of the sample to the melting profiles of the controls10,11.

The EpiMelt Real-Time PCR Master Mix uses the dsDNA intercalating dye EvaGreen® as a reporter. EvaGreen® is well suited for HRM, as it is a stable agent that becomes highly fluorescent when bound to dsDNA, is nonmutagenic and non-cytotoxic, and only has low levels of PCR inhibition13. This makes running HRM under saturating conditions possible, thereby preventing dye redistribution14. The use of EvaGreen® produces robust results with high fluorescence signals and prevents non-specific amplification14,15.

In this application note, we demonstrate robust and sensitive methylation detection using EpiMelt Real-Time PCR Master Mix and the EpiMelt Methylation Detection Assays targeting BRCA1, MLH1, and MGMT.

Materials

Reagents and consumables

EpiMelt BRCA1 Assay (MethylDetect ApS)
EpiMelt MLH1 Assay (MethylDetect ApS)
EpiMelt MGMT Assay (MethylDetect ApS)
EpiMelt Real-Time PCR Master Mix (MethylDetect cat. # EPI – qPCR – 200)
LightCycler® 480 Multiwell plate 96, white (Roche, cat. # 04729692001)

Instruments

LightCycler® 480 System (Roche, cat. # 05015278001)

Methods

Amplification and high-resolution melting

Three EpiMelt Methylation Detection Assays were employed, targeting the promoter regions of BRCA1, MLH1, and MGMT. Each assay contains an assay-specific primer mix, and three assay-specific, ready-to-use controls: a methylated positive control, a methylated negative control, and an assay calibration control. The EpiMelt Real-Time PCR Master Mix is complete, already containing the optimum concentrations of salt and magnesium ions, and no additional preparation or optimization is necessary. For each qPCR reaction, the assay-specific controls were combined with 10.0 μL EpiMelt Real-Time PCR Master Mix 3.0 μL nuclease-free water, and 1.0 μL EpiMelt Primer Mix, according to the manufacturer’s instructions (Table 1). No template controls were included for each assay, and all reactions were run in triplicates.

Table 1: PCR mix.

The PCR amplification and subsequent HRM were performed on the LightCycler® 480 Instrument II in LightCycler® 96-well plates using the setting Detection format » SYBR Green I/HRM dye.

Table 2: PCR and HRM program. *The annealing temperature is assay-specific and may differ between platforms.

For research use only. Not for use in diagnostic procedures. ©2023 MethylDetect. All rights reserved.

The LightCycler® 480 Instrument II PCR amplification and the HRM program are shown in Table 2. The listed annealing temperature is dependent on the assay and the platform. An annealing temperature of 56°C was used for EpiMelt BRCA1, 60°C for EpiMelt MLH1, and 59°C for MGMT. The resulting data were analyzed using the LightCycler® 480 Software.

Results

Using the EpiMelt Methylation Detection Assays and EpiMelt Real-Time PCR Master Mix resulted in efficient amplification and consistent melting profiles after HRM across the three EpiMelt Methylation Detection Assays (Figure 1). This is illustrated by the amplification curves (Figure 1A-C) and the relative signal difference (-d/dT) plots (Figure 1D-F) for each assay.

Figure 1: A-C: Amplification of the controls from (A) EpiMelt BRCA1, (B) EpiMelt MLH1, and (C) EpiMelt MGMT on the LightCycler® 480 Instrument II. D-F: Relative signal difference (-d/dT) plots after HRM for (D) EpiMelt BRCA1, (E) EpiMelt MLH1, and (F) EpiMelt MGMT. The Assay Calibration Control (blue) demonstrates the high sensitivity of the EpiMelt Methylation Detection Assays.

For research use only. Not for use in diagnostic procedures. ©2023 MethylDetect. All rights reserved.

The amplification curves were steep and reproducible for all EpiMelt Methylation Detection Assay replicates, and the Methylation Positive Control has a lower Ct value than the Methylation Negative Control and Assay Calibration Controls. On the relative difference plots, the peaks are uniform. The extraordinary sensitivity of the EpiMelt Methylation Detection Assays is demonstrated here, as the Assay Calibration Control can be separated
from the Methylation Negative Control and identified as methylated (blue curve in Figure 1).

Table 3: Average Tm for the Methylation Negative Control and Methylation Negative Control from the EpiMelt BRCA1, MLH1, and MGMT Assays.

The EpiMelt Real-Time PCR Master Mix included a HotStart DNA Polymerase, which ensured high amplification efficiency, robust results across triplicates, and no no-template amplification.

The use of EvaGreen® DNA intercalating dye in the PCR master mix yielded high fluorescence signals, and highly specific melting profiles, allowing clear distinction between PCR products from methylated positive and methylated negative controls, and detection of DNA methylation with state-of-the-art sensitivity.

Conclusions

Here, we demonstrate the excellent performance of the EpiMelt Real-Time PCR Master Mix in combination with EpiMelt Methylation Detection Assays for robust methylation detection. The MS-HRM results allow easy evaluation of the methylation level of target loci. This application note illustrates the simplicity of methylation analysis with MethylDetect’s ready-to-use EpiMelt Methylation Detection Assays, using high-quality EpiMelt Real-Time PCR Master Mix.

For research use only. Not for use in diagnostic procedures. ©2023 MethylDetect. All rights reserved.

References

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