Sensitive quantitative analysis of murine LINE1 DNA methylation using high resolution melt analysis

Figures & data

Figure 1. Detection of differences in L1 methylation. Melt curve analysis following amplification with the unbiased L1 primers (F_unbiased_mLINE1 and R_unbiased_mLINE1) - (A) the HRM normalized melt curve and (B) the first derivative melt curve (dF/dT), was performed on methylated (red) and unmethylated (green) control DNA as well as a 50% (methylated: unmethylated control) methylated sample (blue).

Figure 2. Detection of 5-aza induced partial demethylation. Vehicle control (DMSO) (blue), 0.125 µM (purple) and 0.5 µM (orange) 5-aza treated A11 cell line genomic DNA was amplified following bisulphite modification with the unbiased L1 primers and subjected along with the methylated (red) and unmethylated (green) controls to melt analysis - (A) the normalized HRM melt curve (n = 3 samples per treatment group is displayed). The samples were then amplified with unmethylated-biased primers. Melt analysis was performed on the PCR products – (B) the normalized HRM curve and (C) the difference graph (one PCR replicate for each of the n = 4–5 samples is displayed); inset is the difference graph with the unmethylated control included.

Figure 3. Quantitation of methylation differences using the Net Temperature Shift. The normalized fluorescence at each temperature point on the melt curve were used to calculate the mean NTS of the 5-aza treated samples for (A) L1, (B) murine B1 family Mm (B1_Mm) and (C) Intracisternal-Particle A Long-terminal Repeat (IAP_LTR) repeat elements (n = 4–5 samples per treatment group) (*p < 10⁻⁵ compared with the vehicle control; + p < 0.05 compared with the 0.125 µM treated 5-aza samples).

Table 2. Frequency of detected variants in L1 target sequence

Sequence Variant	Type	Frequency in alignment of 20 random in silico PCR results	Detected in L1-PCR product DNA sequencing
				1	G > T	10% (T) (2/20)	No
2	G > A	10% (A) (2/20)	Yes
3	t > C	5% (C) (1/20)	No
	t > G	5% (G) (1/20)	Yes
4	G > A	5% (A) (1/20)	No
5	G > A	15% (A) (3/20)	Yes
6	C > T	5% (A) (1/20)	No
7	G > A	10% (A) (2/20)	Yes
8	G > A	30% (A) (6/20)	Yes
9	G > A	40% (G) (8/20)	Yes
10	C > A	5% (A) (1/20)	No

Unmodified genomic DNA specific L1 primers (F_unmod_mLINE1 and R_unmod_mLINE1) were used in an in silico PCR analysis (UCSC Genome). Twenty randomly selected in silico PCR results were aligned and the frequencies of sequence variants detected in the alignment in comparison to the L1-HRM PCR target sequence are shown.

Figure 4. Confirmation of the detection of 5-aza induced demethylation by the L1-assay. (A) The L1 sequence as confirmed by DNA sequencing. The location of sequence variants in the target L1 region (boxed region) in unmodified genomic DNA identified from both sequence analysis of L1-PCR products (amplified with F_unmod_mLINE1 and R_unmod_mLINE1) and in silico PCR analysis (see Table 2) are shown in bold, numbered 1–10 in blue and characterized as an R (A/G), K (T/G) or M (A/C). CpGs are shaded and numbered in black 1–13. The L1-HRM PCR products (n = 4–5 samples per treatment group from n = 1 bisulphite modification and PCR reaction) were then subjected to pyrosequencing. The pyrosequencing primer is highlighted by the green arrow and the region pyrosequenced is shown in green. (B) The percent methylation of each CpG was plotted and separated by 5-aza concentration (vehicle - blue, 0.125 µM - purple and 0.5 µM -orange) or control DNA status (red – methylated control, green – unmethylated control). (C) The mean methylation of all CpGs pyrosequenced (7–12) was plotted vs. 5-aza concentration (*p < 10⁻⁵ compared with the vehicle control; + p < 0.05 compared with the 0.125 µM treated 5-aza samples). (D) Genomic DNA samples (n = 4–5 per treatment group) were subjected to Liquid Chromatography – Mass Spectrometry (LC-MS) and mean percent methylation (5-mdC/dG ratio) for each treatment group plotted (*p < 0.05). (E) Linear regression analysis was performed on the mean NTS for samples of a standard curve (R² = 0.992; p < 0.001). The standard curve was made from mixtures of the vehicle and 0.5 µM 5-aza treated A11 samples (75.6, 75.45, 75.3, 75, 74.09, 72.58, 69.56, 66.54, 63.52, 60.5 and 45.4% methylated) using the mean pyrosequencing values for the two samples. The unmethylated control DNA (~3% methylated) was also included in the L1-HRM PCR reactions. The samples underwent two separate bisulphite modifications and were amplified with the unmethylated-biased primers in two separate PCR reactions per bisulphite modification, prior to melt analysis.

Table 3. Correlation of HRM assay with pyrosequencing and LC-MS

	Pearson Correlation Coefficient
	Treatment	HRM	LCMS	Pyrosequencing (mean methylation)
					Treatment	1	-0.952**	-0.391**	-0.952**
Pyrosequencing (mean methylation)	-0.952**	0.913**	0.377**	1
HRM	-0.952**	1	0.387**	0.913**
LCMS	-0.391**	0.387**	1	0.377**

Analysis of the ability of the L1-HRM assay to detect global DNA demethylation in samples treated with 5-aza when compared with pyrosequencing analysis of L1-HRM PCR products, and LC-MS analysis of genomic DNA methylation (**p < 0.01).

Table 4. L1-HRM inter- and intra-assay variation

Sample Mean Meth (%)	Bisulphite Modification Reaction #1						Bisulphite Modification Reaction #2						Mean NTS	SD
	PCR 1		PCR 2		Mean NTS	SD	PCR 1		PCR 2		Mean NTS	SD
	NTS	SD	NTS	SD			NTS	SD	NTS	SD
45.40	-10.90	0.09	-9.95	0.25	-10.43	0.67	-10.44	0.06	-10.58	0.10	-10.51	0.10	-10.47	0.38
60.50	-7.99	0.07	-7.27	0.11	-7.63	0.51	-7.28	0.08	-7.16	0.01	-7.22	0.08	-7.42	0.36
63.52	-6.87	0.09	-6.26	0.01	-6.57	0.43	-7.40	0.05	-7.08	0.02	-7.24	0.23	-6.90	0.45
66.54	-6.24	0.21	-5.89	0.13	-6.07	0.25	-6.84	0.34	-6.81	0.03	-6.83	0.02	-6.45	0.45
69.56	-5.60	0.08	-4.93	0.00	-5.27	0.47	-5.60	0.06	-5.40	0.09	-5.50	0.14	-5.38	0.30
72.58	-5.64	0.12	-4.89	0.21	-5.27	0.53	-5.11	0.00	-5.11	0.00	-5.11	0.00	-5.19	0.31
74.09	-5.24	0.03	-4.70	0.00	-4.97	0.38	-5.23	0.09	-5.04	0.07	-5.14	0.13	-5.10	0.20
75.00	-5.25	0.03	-4.03	1.01	-4.64	0.86	-4.78	0.05	-4.63	0.03	-4.71	0.11	-4.67	0.60
75.30	-4.71	0.07	-4.68	0.13	-4.70	0.02	-4.74	0.03	-4.64	0.08	-4.69	0.07	-4.69	0.07
75.45	-5.05	0.06	-4.67	0.01	-4.86	0.27	-4.73	0.04	-4.86	0.04	-4.80	0.09	-4.83	0.16
75.60	-5.09	0.03	-4.03	0.72	-4.56	0.75	-4.70	0.06	-4.76	0.16	-4.73	0.04	-4.66	0.50

The 5-aza standard curve made from mixtures of the vehicle and 0.5 µM 5-aza treated A11 samples (75.6, 75.45, 75.3, 75, 74.09, 72.58, 69.56, 66.54, 63.52, 60.5 and 45.4% methylated) using the mean pyrosequencing values for the two samples, was used to assess intra- and inter-assay variability. Each sample underwent two separate bisulphite modifications and two separate HRM-PCR runs with two PCR reaction duplicates per HRM-PCR.

Figure 5. Detection of tissue and sex specific methylation levels in murine tissues. (A) The mean NTS of L1 elements was used to assess DNA methylation levels in the kidney, liver, peripheral blood (PB), prostate and spleen tissues of male (n = 5) and female (n = 5) Balb/c and CBA mice following bisulphite modification and amplification with the unmethylated-biased primers. (B) The mean NTS of the kidney, liver, PB, prostate and spleen of male Balb/c mice when amplified with the unbiased primers for comparison with the unmethylated-biased primers.

Table 1. Primer sequences

Primer Biasing	Primer	Sequence	Number of CpGs in target sequence	Amplicon Size
						F_kato	TAG GAA ATT AGT TTG AAT AGG TGA GAG GGT
unbiased	F_unmod_mLINE1	GGG CTG AGG CAG CAC CCT GTG TG	13	195 bp
	R_unmod_mLINE1	TCC AGA AGC TGT CAG GTT CTC TGG C
	F_unbiased_mLINE1*	GGT TGA GGT AGT ATT TTG TGT G		195 bp
	R_unbiased_mLINE1	TCC AAA AAC TAT CAA ATT CTC TAA C
unmethylated	F_umod_unmeth_mLINE1	GCT GAG GCA GCA CCC TGT GTG GGC C	11	193 bp
	R_umod_unmeth_mLINE1	TCC AGA AGC TGT CAG GTT CTC TGG CGC
	F_unmeth_mLINE1*	GTT GAG GTA GTA TTT TGT GTG GGT T		193 bp
	R_unmeth_mLINE1	TCC AAA AAC TAT CAA ATT CTC TAA CAC
	R_pyroprimer	TAT TCAA ACT AAT TTC CTA A
	F_unmod_unbiased_B1_Mm	AGC CGG GCG TGG TGG	7	92 bp
	R_umod_unbiased_B1_Mm	CTT TGT AGA CCA GGC TGG CCT C
	F_mod_unbiased_B1_Mm	AGT YGG GYG TGG TGG		92 bp
	R_mod_unbiased_B1_Mm	CTT TAT AAA CCA AAC TAA CCT C
	F_unmod _unmeth_IAP_LTR	CCA CAT TCG CCG TTA CAA GAT GGC	6	141 bp
	R_unmod _unmeth_IAP_LTR	CAC CTA AAA ACA TAT CAC T
	F_mod _unmeth_IAP_LTR	TTA TAT TTG TTG TTA TAA GAT GG T		141 bp
	R_mod _unmeth_IAP_LTR	CAC CTA AAA CAT ATC ACT

All primers were designed using MethPrimer and UCSC Genome Browser. Bold typeface indicates bisulphite modified bases and biased CpG sites are underlined. Legend Key: unbiased = unbiased for methylated or unmethylated bisulphite modified genomic DNA; unmeth = unmethylated-biased primers; unmod = unmodified genomic DNA specific primers. B1_Mm = mouse B1 element, family Mm; IAP_LTR = Intracisternal-A-particle Long-terminal Repeat element. *Primers contain a 5′ biotin tag.