Epigenetics and High ResolutionMelting-PCR7th European Elastin Meeting1st -4thseptember 2012Giulia Annovi, PhDUniversity o...
HistonemodificationsDNAmethylationEpigenetic mechanisms are able to produce differentphenotypes from a single genotypeEffe...
MethPrimer analysis of promoters : http://www.urogene.org/methprimer/index1.htmlNCBI Epigenomics: http://www.ncbi.nlm.nih....
The bisulfite conversionHigh Resolution Melting (HRM)Tissue samplesfixed in paraffinCell culturess-DNADNA extraction
High Resolution Melting (HRM) - PCR=> Melting curves allow discrimination between methylated and unmethylatedsamples, beca...
100% Methylated0% Methylated100% MethylatedUnconvertedQuantitation data forCycling:- converted ssDNA integrity- inter- and...
⇒Global Melt Curves analysis can’t give a clear result: cytosines methylation inhuman samples is unbalanced and depends on...
HRM is a powerful technique to identify methylation at specificpromoter very quickly and from different kind of samples.pR...
HRM is a powerful technique to identify methylation at specificpromoter very quickly and from different kind of samples.74...
Thank you for your attention!
DNMT1DNMT3A, DNMT3BDNA methyl-transferase: transfers a methyl group to DNA
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Annovi HRM and Epigenetics

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  • Because it is implicated in the organization of chromatin structure and regulation of gene transcription
  • activity of DMNTs, a class of enzimes catalazying the transfer of a methyl moiety to cytosines principally in the CpG dinucleotide.
  • Annovi HRM and Epigenetics

    1. 1. Epigenetics and High ResolutionMelting-PCR7th European Elastin Meeting1st -4thseptember 2012Giulia Annovi, PhDUniversity of Modena and Reggio Emilia, ItalyIBCP Lyon, FranceMoulin L., Cenizo V., André V., Sommer P., Debret R.
    2. 2. HistonemodificationsDNAmethylationEpigenetic mechanisms are able to produce differentphenotypes from a single genotypeEffect of DNA methylation:# transcriptional repression# compact chromatinestructures# normal embryologicaldevelopment# X chromosome inactivation# imprinting controlO NH2CH3Cytosine
    3. 3. MethPrimer analysis of promoters : http://www.urogene.org/methprimer/index1.htmlNCBI Epigenomics: http://www.ncbi.nlm.nih.gov/epigenomics/view/genomeSize (Start - End)Island 1 401 bp (71 - 471)Island 2 261 bp (571 - 831)Predicted CpG islands
    4. 4. The bisulfite conversionHigh Resolution Melting (HRM)Tissue samplesfixed in paraffinCell culturess-DNADNA extraction
    5. 5. High Resolution Melting (HRM) - PCR=> Melting curves allow discrimination between methylated and unmethylatedsamples, because melting temperature is nucleotides composition dependent.
    6. 6. 100% Methylated0% Methylated100% MethylatedUnconvertedQuantitation data forCycling:- converted ssDNA integrity- inter- and intra-samplecomparable amplification100% Methylated0% MethylatedHRM Data analysis
    7. 7. ⇒Global Melt Curves analysis can’t give a clear result: cytosines methylation inhuman samples is unbalanced and depends on environnement.⇒ We have to analyse each curve independently to identify melting temperature foreach sample.100% Methylated0% Methylated100% Methylated
    8. 8. HRM is a powerful technique to identify methylation at specificpromoter very quickly and from different kind of samples.pRloxl1 pRfbn1 pRfbln5Predicted CpG islands-Moulin L. Romain D.-
    9. 9. HRM is a powerful technique to identify methylation at specificpromoter very quickly and from different kind of samples.74.9575.0575.1575.2575.3575.4575.55a-SMA promoter methylationCollagen5AZAMeltTemp=Methylationlevel00.511.522.533.544.55a SMA expression - qPCRFibro 5AZAmRNAaSMAexpressionFibroblasts Fibroblasts + 5 AZA
    10. 10. Thank you for your attention!
    11. 11. DNMT1DNMT3A, DNMT3BDNA methyl-transferase: transfers a methyl group to DNA

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