1. Epigenetics and High Resolution
Melting-PCR
7th European Elastin Meeting
1st -
4th
september 2012
Giulia Annovi, PhD
University of Modena and Reggio Emilia, Italy
IBCP Lyon, France
Moulin L., Cenizo V., André V., Sommer P., Debret R.

2. Histone
modifications
DNA
methylation
Epigenetic mechanisms are able to produce different
phenotypes from a single genotype
Effect of DNA methylation:
# transcriptional repression
# compact chromatine
structures
# normal embryological
development
# X chromosome inactivation
# imprinting control
O NH2
CH3
Cytosine

3. MethPrimer analysis of promoters : http://www.urogene.org/methprimer/index1.html
NCBI Epigenomics: http://www.ncbi.nlm.nih.gov/epigenomics/view/genome
Size (Start - End)
Island 1 401 bp (71 - 471)
Island 2 261 bp (571 - 831)
Predicted CpG islands

4. The bisulfite conversion
High Resolution Melting (HRM)
Tissue samples
fixed in paraffin
Cell culture
ss-DNA
DNA extraction

5. High Resolution Melting (HRM) - PCR
=> Melting curves allow discrimination between methylated and unmethylated
samples, because melting temperature is nucleotides composition dependent.

6. 100% Methylated
0% Methylated
100% Methylated
Unconverted
Quantitation data for
Cycling:
- converted ssDNA integrity
- inter- and intra-sample
comparable amplification
100% Methylated
0% Methylated
HRM Data analysis

7. ⇒Global Melt Curves analysis can’t give a clear result: cytosines methylation in
human samples is unbalanced and depends on environnement.
⇒ We have to analyse each curve independently to identify melting temperature for
each sample.
100% Methylated
0% Methylated
100% Methylated

8. HRM is a powerful technique to identify methylation at specific
promoter very quickly and from different kind of samples.
pRloxl1 pRfbn1 pRfbln5
Predicted CpG islands
-Moulin L. Romain D.-

9. HRM is a powerful technique to identify methylation at specific
promoter very quickly and from different kind of samples.
74.95
75.05
75.15
75.25
75.35
75.45
75.55
a-SMA promoter methylation
Collagen
5AZA
MeltTemp=Methylationlevel
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
a SMA expression - qPCR
Fibro 5AZA
mRNAaSMAexpression
Fibroblasts Fibroblasts + 5 AZA

10. Thank you for your attention!

11. DNMT1
DNMT3A, DNMT3B
DNA methyl-transferase: transfers a methyl group to DNA