Determining methylation status of methylguanine DNA methyl transferase (MGMT) from formalin-fixed, paraffin embedded tumor tissue

Graphical abstract


Specimen requirements
This assay is performed on tumor DNA isolated from FFPE tissue or fresh/frozen tissue. Quantity, purity and quality of extracted nucleic acids should be assessed by traditional methods before proceeding with this protocol. Appropriate block(s) or tissue specimens should be selected by the pathologist who reviewed the case. FFPE tissue blocks used should contain greater than 50% tumor tissue when non-tumor tissue is also present. If such a block is not available, tumor tissue can be dissected from an unstained section on a glass slide. Performance of this assay is compromised when lower quantities of tumor cells are used as the methylation status of tumor cells is diluted out by methylation status of normal cells.
Note: Sections fixed in a heavy metal fixative or specimens that have been decalcified are not acceptable for this procedure. Tissues fixed in heavy metal fixatives are known to inhibit downstream PCR amplification while decalcification of tissues is known to significantly degrade genomic DNA.

Major equipment
Bio-Rad DNA Engine 1 Thermal Cycler (PTC-200) ( Quality control/process control Positive Control. Each run must contain two positive controls that consist of methylated DNA. The CpGenome Universal Methylated DNA (Millipore) has to be diluted to 10 ng/uL for optimal results and bisulfite converted. The Converted Methylated DNA (Promega) needs to be diluted to 3 ng/mL. Negative Control: Each run must contain one negative control that consists of unmethylated DNA. The CpGenome Universal Unmethylated DNA (Millipore) has to be diluted to 10 ng/mLfor optimal results and bisulfite converted. No Template Control (NTC). Each run must contain a no template control (NTC) that consists of all reagents with the exception of any DNA sample.
Note: The bisulfite conversion of the controls must be done at the same time as the samples.
DNA conversion: MethylEdge Bisulfite Conversion System (Promega) [2] All reagents are stable at room temperature a. Prepare 1X ME Wash Buffer Add 24 mL of 95-100% ethanol to the bottle containing 6 mL of concentrated ME Wash Solution. Mark on the bottle that you have performed this step. b. Prepare Samples In a 0.2 mL PCR tubes, dilute 50 ng of each sample and control DNA in a total volume of 20 mL.
Assay performance was optimized for 50 ng DNA because in small tissue biopsies large quantities of DNA may not be obtained.
If the sample volume is less than 20 mcl, adjust the volume to 20 mcl with nuclease-free water. You should prepare 3 tubes of the methylated DNA and 2 of the unmethylated DNA.

c. Bisulfite Conversion
Add 130 mcl of Bisulfite ME Conversion Reagent to each DNA sample and control, and pipet ''up and down'' to mix. Centrifuge briefly to collect the sample at the bottom of the tube.
Ensure that the cap of the ME Conversion Reagent is closed tightly before storing the remaining reagent. Perform the conversion reaction using the cycling parameters shown in Table 1 on the Bio-Rad DNA Engine 1 Thermal Cycler (Vf = 150 mcl).
According to the Manufacture's protocol, samples can be storage at 4 8C or on ice (protected from light) for up to 20 h. For each sample to be processed, place a ME Spin Column into one of the Collection Tubes. Add 600 mcl of ME Binding Buffer to the ME Spin Column. Transfer the entire bisulfite-treated sample to the column. Invert the tube several times ($15Â). Spin at maximum speed !10,000 Â g for 30 s. Discard the flow through and re-insert the ME Spin Column into the same collection tube.
Add 100 mcl of 1Â ME Wash Buffer (with ethanol added). Spin at maximum !10,000 Â g for 30 s. Add 200 mcl of ME Desulphonation Buffer to each ME Spin Column. Incubate at room temperature for 15 min. Spin at maximum !10,000 Â g for 30 s. Add 200 mcl of ME Wash Buffer (ethanol added). Spin at maximum !10,000 Â g for 30 s. Repeat this wash step once more. Place the ME Spin Column into a clean 1.5 mL microcentrifuge tube. Add 15 mcl of ME Elution Buffer. Incubate for 1 min at room temperature. Spin at maximum !10,000 Â g for 30 s. Remove and discard the ME Spin Column. Store the DNA at À20 8C (protected from light).
Pool the CpGenome Universal Methylated DNA that was converted (total of 3 tubes) into one tube Pool the CpGenome Universal Unmethylated DNA that was converted (total of 2 tubes) into one tube MethyLight reaction set-up [3] Thaw all reagents on ice, vortex and briefly centrifuge all reagents before dispensing. Prepare 3 ng/mcl dilution of the Converted Methylated DNA (Promega) (dilution can be stored in the À20 8C freezer).  One PCR reaction must be run for each sample to be tested.
a. In 5 clean labeled 0.2 mL PCR tubes, make 5 serial dilutions 1:5 of the converted CpGenome Universal Methylated DNA as shown in Table 2. CpGenome Universal Methylated DNA was converted in the previous step. b. In two clean labeled 1.5 mL microcentrifuge tube prepare the MGMT and ACTB master mixes as shown in Table 3  Verify that the reaction volume is set to 20 mcl. Verify the thermal profile, as shown in Table 5, and edit if necessary. Click on the ''Start Run'' icon on the top right hand corner of the screen. Be sure to save the run as a unique file name.

Data analysis
When the run has completed, the software should automatically open the ''Analysis'' window.
In the Analysis window with all samples selected, verify the efficiency of each standard curve for MGMT and ACTB. The value should be between 90 and 110%. Verify the threshold already established by the software. Make sure that all samples and controls have the same threshold.  The threshold should be in the middle of the exponential curve.
Then click on the ''Analysis'' icon on the left hand side of the screen. Export the results to an excel file.
Select ''Export'', then ''Browse'' ! ''Desktop'' ! ''MGMT'' folder. After that, select ''Start Export''. The excel file with the data will be exported to the Ion folder, and then you can close the window.
Save the excel file generated by the software. Copy the ''t'' and ''Quantity mean'' values of each sample from the ''Analysis'' file and paste them onto the ''MGMT Worksheet''. Automatically the PMR (Percentage of Methylated Reference) will be calculated. If the ACTB of a specific sample does not amplify, the sample must be repeated. It cannot be called unmethylated.