254
of bisulfite-converted DNA, 2 μl of RNase free water, and 1 μl
of forward and reverse primers (see Note 5).- Run the PCR with thermal profile of 15 min at 95 °C, fol-
lowed by 50 cycles of 30 s at 95 °C, 30 s at 61 °C, and 20 s at
72 °C (see Note 6). - Generate the melt curve by increasing temperature from 70 to
95 °C for all reactions, with an increase rate of 0.05 °C/s. - Analyze the melt curve with appropriate software and deter-
mine the methylation status of particular gene of interest. - Check the purity of MS-HRM product in 1.5% agarose gel (see
Note 7). - Purify the PCR products and proceed for sequencing as men-
tioned in previous section.
4 Notes
- Freshly dissolve 7.49 g sodium bisulfate in 15 ml double dis-
tilled water and mix for 10 min with protection from light.
Then adjust the pH to 5.0 using NaOH. - Dissolve 22 mg hydroquinone in 10 ml double distilled water
with inversion. - The volume of 3 N NaOH is exactly 1/10th the final volume.
- This step is to add the glycogen as the carrier for the precipita-
tion, which is initiated by the ammonium acetate. - Template DNA and primers concentrations need to be opti-
mized for each specific gene of interest. - The thermal profiles and reaction cycle also need to be opti-
mized for each individual targets. Usually, for bisulfite-con-
verted DNA more reaction cycle would generate products that
are more precise. - For effective determination of DNA methylation with
MS-HRM, the products need to be pure. Impurities or multi-
ple bands in gel of MS-HRM could indicate misleading DNA
methylation status. Optimization of thermal profiles or primers
pairs could give pure MS-HRM products.
Acknowledgements
Farhadul Islam and Johnny C. Tang contributed equally to this
work as first authors.Farhadul Islam et al.