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Methylome and epigenetic markers
Published in Moshe Hod, Vincenzo Berghella, Mary E. D'Alton, Gian Carlo Di Renzo, Eduard Gratacós, Vassilios Fanos, New Technologies and Perinatal Medicine, 2019
Skevi Kyriakou, Marios Ioannides, George Koumbaris, Philippos Patsalis
Currently there are three main approaches for the detection and analysis of methylation patterns across the genome. First, utilizing methylation-sensitive restriction digestion enzymes, the enzyme's restriction site is used for enrichment of specific methylated regions. This approach has two main drawbacks: it is limited to the regions containing the restriction sites, and it is highly susceptible to false positive results due to incomplete digestion of the DNA (9). The second approach, sodium-bisulfite–based treatment, involves the conversion of unmethylated cytosines to uracils leaving the methylated cytosines unchanged. Bisulfite conversion is regarded as the gold standard for DNA methylation analysis since it enables mapping of methylated sites at the single base pair resolution (10). However, due to the chemical treatment of DNA, it can cause substantial degradation of DNA and incomplete conversion of unmethylated cytosines to uracils leading to false positive results (11). The third approach involves the enrichment of methylated DNA through affinity-based assays, involving the use of antibodies specific to methyl sites, namely, methylated DNA immunoprecipitation (MeDIP), or the use of methyl-binding proteins (MBD) (12,13).
Epigenetics
Published in Sara C. Zapico, Mechanisms Linking Aging, Diseases and Biological Age Estimation, 2017
Christian Thomas, Sara C. Zapico
The number of techniques to analyze the epigenetic changes in the genome is constantly growing. For DNA methylation, bisulfite DNA treatment followed by clonal sequencing has been traditionally applied. However, the increased availability of genome-wide approaches such as microarray or deep sequencing have allowed for high throughput analyses. Based on bisulfite conversion, BeadArray (Illumina) platform (with bisulfite conversion) analyzes a moderate number of CpGs across the genome in a large number of samples (Fouse et al. 2010). Another strategy has been the use of microarrays with methylation-sensitive and—insensitive restriction endonuclease digestion or affinity-based enrichment of methylated DNA by Chromatin Immunoprecipitation (ChIP), also known as Methylated DNA Immunoprecipitation (MeDIP) (Ben-Avraham et al. 2012, Qureshi and Mehler 2011). Next-Generation Sequencing (NGS) based methods, mostly linked to ChIP (MeDIP-seq) but also linked to Bisulfite Treatment (BS-seq), have been established for detection of DNA methylation across the entire genome, developing a variety of platforms to increase the number of CpGs for which methylation can be assessed (Ben-Avraham et al. 2012).
Site specific hypermethylation of CpGs in Connexin genes 30, 26 and 43 in different grades of glioma and attenuated levels of their mRNAs
Published in International Journal of Neuroscience, 2019
Jayalakshmi J., Arambakkam Janardhanam Vanisree, Shantha Ravisankar, Rama K.
To further ascertain the observed methylation status of the promoter and intron region of gene of interest, enrichment of methylated DNA was carried out by Methylated DNA immunoprecipitation (MeDIP) assay. To validate the MeDIP assay, qRT-PCR was done in the immunoprecipitated DNA as well as input DNA and the enrichment of methylated DNA was expressed as Input %. Connexin 30 had exhibited a significant enrichment in grade II (p < 0.02), grade III and IV (p < 0.01) (Figure 6a) when compared to control, but no significant changes were observed in grade I (Pilocytic astrocytoma). Unlike Connexin 30, Connexin 26 showed significant increase in the enrichment among all the grades (grade I p < 0.02, grade II–IV p < 0.03) with respect to control (Figure 6b). On the contrary, Cx43 (Figure 6c) could exhibit significance in its enrichment only in grade IV (p < 0.05) when compared to control.
Liquid biopsy from research to clinical practice: focus on non-small cell lung cancer
Published in Expert Review of Molecular Diagnostics, 2021
Umberto Malapelle, Pasquale Pisapia, Alfredo Addeo, Oscar Arrieta, Beatriz Bellosillo, Andres F. Cardona, Massimo Cristofanilli, Diego De Miguel-Perez, Valeria Denninghoff, Ignacio Durán, Eloísa Jantus-Lewintre, Pier Vitale Nuzzo, Ken O’Byrne, Patrick Pauwels, Edward M. Pickering, Luis E. Raez, Alessandro Russo, Maria José Serrano, David R. Gandara, Giancarlo Troncone, Christian Rolfo
The development of monoclonal antibodies specific to 5-methylcytosines revolutionized the analyses of DNA methylation, providing an unprecedented opportunity to map and compare complete DNA CpG methylomes [91,97]. The limitation of the methods based on affinity enrichment, including methylated DNA immunoprecipitation sequencing (MeDIP-seq) assay, was that large amount of cfDNA were needed. The new method called cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-Seq) showed a detection of methylated ctDNA with high sensitivity and specificity, even at low sequence depth and using 1–10ng of cfDNA [91,92].
Splicing deregulation, microRNA and notch aberrations: fighting the three-headed dog to overcome drug resistance in malignant mesothelioma
Published in Expert Review of Clinical Pharmacology, 2022
Dario P. Anobile, Giulia Montenovo, Camilla Pecoraro, Marika Franczak, Widad Ait Iddouch, Godefridus J Peters, Chiara Riganti, Elisa Giovannetti
Kim et al. evaluated the epigenetic mechanisms involved in the tumor cell heterogeneity using side population (SP) and non-SP cells isolated from MS-1, a human malignant mesothelioma cell line [70]. The cancer cells were analyzed by methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) and RNA-seq methodology. They identified changes in DNA methylation in 122 genes, of which 118 genes were hypermethylated and thus downregulated and four genes were hypomethylated and thus upregulated.