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Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

Artikel i vetenskaplig tidskrift
Författare Santhilal Subhash
Meena Kanduri
Publicerad i Journal of Visualized Experiments
Volym Genetics
Nummer/häfte 124
ISSN 1940-087X
Publiceringsår 2017
Publicerad vid Institutionen för biomedicin, avdelningen för klinisk kemi och transfusionsmedicin
Institutionen för biomedicin, avdelningen för medicinsk kemi och cellbiologi
Språk en
Länkar https://doi.org/10.3791/55773
https://www.jove.com/video/55773
Ämnesord DNA methylation, methyl CpG DNA binding domain protein, differentially methylated regions, next-generation sequencing, protein coding and noncoding genes, chronic lymphocytic leukemia
Ämneskategorier Cell- och molekylärbiologi

Sammanfattning

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.

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