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Differential DNA Methylation Patterns in Endo-siRNAs Mediated Silencing of LINE-1 Retrotransposons

  • Long Chen
  • Jane E Dahlstrom
  • Danny RangasamyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1173)

Abstract

Analyzing differences in DNA methylation is a powerful tool for assessing the effect of endo-siRNAs expression in the human genome. Here, we present a simple genome-wide DNA methylation assay that allows for a precise quantitative analysis of differences in the promoter of human long interspersed nuclear element 1 (LINE-1 or L1) retrotransposons in response to endogenous and exogenous expression of endo-siRNAs. Using the DNA bisulfite modification sequencing, we have optimized the method to detect small changes in heterogeneously methylated L1 repeats at multiple regions across the genome. We also provide guidance for analysis of primary bisulfite sequencing data and interpretation of the methylation status using the Web-based bisulfite sequencing DNA methylation (BISMA) analysis. This refined and reproducible assay can be performed even using a small amount of genomic DNA and is suitable for the analysis of clinical tissue samples.

Keywords

Endo-siRNAs LINE-1 Breast cancer cells DNA methylation DNA sequencing PCR Bisulfite data analysis 

Notes

Acknowledgments

This work was supported by a grant from the Canberra Hospital Private Practice Fund and the ACT Health and Medical Research Support Program.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Long Chen
    • 1
  • Jane E Dahlstrom
    • 1
    • 2
  • Danny Rangasamy
    • 1
    Email author
  1. 1.John Curtin School of Medical ResearchThe Australian National UniversityCanberraAustralia
  2. 2.Department of Anatomical PathologyThe Canberra HospitalGarranAustralia

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