The JAX MGMT Promoter Methylation Assay utilizes High Resolution Melt (HRM) analysis of DNA for MGMT Promoter Methylation detection in CNS tumors
Test Overview
MGMT promoter methylation status is "an essential part of molecular diagnostics for all high-grade gliomas" as stated by NCCN guidelines. The methylation status is a predictor for TMZ treatment in CNS tumors, as well as a prognostic marker. For The Jackson Laboratory Clinical Genomics Laboratory, we aim to establish CNS tumor center of excellence by the collaboration of JAX researchers and local clinicians. MGMT methylation testing is critical for CNS tumors.
Test Description
The JAX MGMT Promoter Methylation Assay utilizes High Resolution Melt (HRM) analysis of DNA for MGMT Promoter Methylation detection in CNS tumors.
Methods
The JAX MGMT Promoter Methylation Assay utilizes a quantitative PCR (qPCR) followed by high-resolution melt analysis (HRM) to identify MGMT promoter methylation. Genomic DNA is extracted from FFPE tissues (minimum 30% neoplastic content) and bisulfite treated using the EZ DNA Methylation-Gold Kit (Zymo). The bisulfite-treated DNA is amplified via qPCR followed by a melting analysis on an Applied Biosystems QuantStudio 7. The area under the curve (AUC) is calculated for the HRM derivative plots for both methylated and unmethylated peaks and the ratio of methylated to unmethylated is calculated. Specimen will be interpreted as MGMT Promoter Unmethylated if the methylated/unmethylated ratio falls within the validated unmethylated range. Specimen with ratios above a 15% methylated control will be interpreted as MGMT Promoter Methylated and specimen with ratios between unmethylated and 15% will be interpreted as Indeterminate.
Specimen Requirements:
Clinical Sample Acceptance Criteria
- FFPE Slides - uncoated unbaked slides in plastic slide containers
- 1 H&E slide and 10 adjacent unstained 5um sections. Area of tumor cell content should be a minimum of 3x3mm or 5,000 cells and be comprised of at least 30% cancer cells.
- FFPE Block - send in sealed biohazard bag
- Area of tumor cell content should be a minimum of 3x3mm or 5,000 cells and be comprised of at least 30% cancer cells.
- DNA:
- Sample Concentration: ≥ 10 ng/uL
- Sample Quality: < 5 (▲CT)
- Amount of Material: ≥ 200 ng
Samples that don’t meet the above acceptance criteria may fail to generate useable data.
Please deliver samples to:
The Jackson Laboratory for Genomic Medicine
Attn: Clinical Genomics Laboratory
10 Discovery Drive
Farmington, CT 06032
Contact Us:
860-837-2320
CGL_CS@jax.org