2.6 | Measuring methylation rate
The 20 µl of 300 ng extracted DNA samples (section 2.3) and purified
standard DNA (section 2.4) were subjected to bisulfite conversion using
the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA). This
converts unmethylated cytosines to uracil. The differences between the
bisulfite-converted DNA samples were measured using MS-HRM using a
LightCycler 480 Instrument II (Roche Molecular Systems, Branchburg, NJ,
USA) equipped with the Gene Scanning Software in a 25 µl total volume
containing 1x EpiTect HRM PCR Master Mix (EpiTect HRM PCR kit: QIAGEN
GmbH, Hilden, Germany), 750 nM of each primer, and 2–4 µl of template
DNA (bisulfite-converted DNA). The chosen detection format was set to
SYBR Green I/HRM Dye. First, the initial PCR activation step was
performed at 95 ˚C for five mins to activate the polymerase, then the
three-step PCR was performed (PCR conditions are presented in Table 1).
Finally, MS-HRM analysis was initiated by cooling the samples to 65 ˚C
for 1 sec and then heated to 95 ˚C at a ramp rate of 0.02 ˚C/s. Gene
Scanning Software was used to normalize the raw melt curves, allowing
the comparison of samples. After MS-HRM, the normalizing process was
performed, the pre-melt temperature region was set to 72–74 ˚C and
post-melt temperature region to 82-84 ˚C for both gene.
We defined the minimum absolute value of the relative signal difference
from the difference plots as the Df value. For the analysis, we used the
average Df value, which was calculated by measuring each sample twice.
To construct a standard curve that correlates the Df value with the
methylation rate, we used the 100% and 0% methylated standard DNA
samples described in section 2.2. These standard DNA samples were mixed
in various ratios, resulting in a set of standard DNA samples with
different methylation percentages (0, 5, 10, 15, 20, 50, and 100%). The
Df values of the standard curve were obtained from the MS-HRM using the
standard DNA samples for both genetic regions. The plotted standard
curve followed a non-linear regression model designed by Wameckeet al. (1997ed by Hamano et al . (2016) as follows:
\begin{equation}
\frac{a*M}{b-M}=\ \frac{\text{Df}}{\text{Df}_{\max}-\ Df}\nonumber \\
\end{equation}Where a: coefficient, b: The maximum methylation rate used
for the standard curve construction, M: methylation rate,\(\text{Df}_{\max}\): Df value of the maximum methylated standard
samples (GRIA2: 0.2, CDKN2A: 1.0).
Due to the low range of methylation percentages observed in GRIA2during subsequent analysis, the standard curve was redrawn using the
standard DNA samples with the methylation percentages of 0, 5, 10, 15,
and 20%. All subsequent analyses of GRIA2 utilized this
recalibrated calibration curve to quantify the methylation percentage of
the samples.