High-resolution melting point analysis technique for detection of methylation in frozen tissue samples

Roche Cancer Research Application: High-resolution Melting Point Analysis for Detection of Methylation in Frozen Tissue Samples
1 Introduction
Promoter hypermethylation is a very common phenomenon in the transcription of malignant tumor genes and is considered as one of the hallmarks of malignant transformation of cells. DNA methylation analysis is a genetic testing tool for early cancer detection, risk assessment and therapeutic efficacy assessment with very attractive prospects.
The most common method for DNA methylation detection relies on the treatment of genomic DNA by sodium bisulfite, converting cytosine to uracil and unmodified 5-methylcytosine. The modified gene sequence is altered to identify methylated cytosine in subsequent PCR amplification products.
Through sulfite sequencing, the most accurate assessment of gene methylation can be performed to identify monomethylated cytosine. Several simpler PCR-based methods have been developed that are important for small-scale research laboratories. These methods are newer, simpler, and easier to operate. High resolution melting point analysis (HRM) is one of several methods. HRM is based on the "melting" nature of DNA in solution. The principle of the method is to distinguish disulfate-treated DNA templates with different methylated cytosine contents by melting point analysis based on different melting temperatures. HRM is a relatively simple and relatively inexpensive method because it does not require expensive probes and standardization of the reference gene. Through the application of HRM technology, all CpGs in the amplicon can be analyzed, and the homology and heterologous methylation can be distinguished according to the shape of the melting curve. This is very important because the methylation pattern of the promoter CpG island is usually non-homology.
Frozen tissue is a very important resource for biomarker detection. The performance of the main analytical methods was tested on formalin-fixed, paraffin-embedded (FFPE) tissue. The purpose of the current study was to evaluate and confirm the HRM analysis of promoter methylation in FFPE tissues from individuals with colorectal cancer.
2 Materials and methods
2.1 Control and study samples
CpGenome universal methylated DNA (Chemicon/Millipore, Billerica, MA) and DNA from peripheral blood mononuclear cells (PBMNCs) were used as methylation and non-methylation controls. Methylation control DNA was diluted in unmethylated PBMC DNA to generate methylation standards (50%, 25%, 10%, 5%, 1%, and 0.1%). After optimization of HRM testing, we analyzed frozen tissue samples from individuals with colorectal tumors.
2.2 DNA extraction and disulfate modification
PBMNC DNA and DNA from healthy volunteers from cultured tumor cell lines were isolated. For DNA isolates from cryopreserved tissue, sections of 10 μm thickness from FFPE tumor tissue blocks were used.
2.3 MethyLight detection
For a description of the MGMT and APC MethyLight tests, see the previous literature description. Briefly, PCR was performed on a LightCycler® 480 instrument.
2.4 high resolution melting point analysis
PCR amplification and HRM were performed on a LightCycler® 480 instrument (Roche) . Considering that FFPE DNA is highly fragmented, large amplicons cause a decrease in melting point resolution, and the amplicon is designed to not exceed 200 bp.
HRM data analysis was performed using Gene Scanning and the TM Calling software module (Roche). The melting curve is normalized by calculation of two normalized regions representing the pre-melting and post-melting of the PCR product. By this calculation, direct comparisons can be made to samples with different starting fluorescence intensities. The normalized temperature conversion melting curve shows that as the temperature increases, the fluorescence intensity decreases.
3 results and discussion
Standard dilutions were tested to determine the sensitivity of HRM detection. With all HRM assays (APC, MGMT, GSPT1 and PTEN), we were able to detect 1% methylated DNA in a non-methylated DNA background based on guaranteed reproducibility (Figure 1A-D) .

Figure 1 : Sensitivity of MGMT ( A ), APC ( B ), GSTP1 ( C ) and PTEN ( D ) methylation HRM detection.

The methylation standard curve is displayed in three ways. 100% methylation is shown in red, 50% methylation is shown in orange, 25% methylation is shown in green, 10% methylation is shown in cyan, 1% methylation is shown in yellow, 0.1% methyl The display is purple and 0% methylation is shown in blue.

Next, we evaluated the effect of formalin-fixed, wax-embedded treatment on promoter hypermethylation testing. We were able to detect the high methylation of the APC promoter with high detection reliability. Figure 2 (A–C) shows that the reproducibility and consistency of fresh and FFPE cell line DNA APC HRM assays are similar.

Figure 2: Using different tumor cell lines - methylation standards (black curve, 100%, 50%, 25%, 10%, 1%, 0.1%, 0%) and samples (color curves) Triple display. The methylation standard (black curve, 100%, 50%, 25%, 10%, 1%, 0.1%, 0%) and the sample (color curve) are shown in triple. (C) Inter-assay variability of fresh and FFPE tumor cell line DNA APC HRM assays from 8 breast and prostate cancer cell lines. The bar chart represents the mean and standard deviation of the six independent experiments.
The FFPE tissue promoter HRM methylation analysis has the following potential applications. The determination of methylation of primary tumors with lymph nodes and distant metastases not only elucidates epigenetic events that occur during disease progression, but also aids in the identification of predictive markers for paraffin-embedded tissue samples.
4 conclusion
Our study further confirmed the applicability of FFPE tissue promoter methylation analysis to quantify HRM. FFPE tissue is the largest source of sample material for normal controls and diseased tissues, and the application of these tissues is invaluable for research. Methodological evaluation is very important for the confirmation of experimental stability and can help to establish whether the test method is suitable as a research tool and possible routine detection means.
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