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Detection of 5-Methylcytosine in Specific Poly(A) RNAs by Bisulfite Sequencing

  • Thomas Amort
  • Alexandra LusserEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1562)

Abstract

RNA bisulfite sequencing (RNA-BS-seq) represents a method for the detection of methylated cytosines in RNA. Developed originally for the analysis of DNA methylation, a modified version of this method can be used for the analysis of methylated cytosine in RNA. Treatment of nucleic acids with HSO3-ions under acidic conditions results in deamination of cytosine (C) to uracil, while 5-methylcytosine (m5C) or 5-hydroxymethylcytosine (hm5C) exhibit low reactivity in this reaction and remain unchanged. Subsequent PCR amplification and sequencing of specific targets allows for the assessment of the methylation status of single Cs in their native sequence context at nucleotide resolution. Here, we describe the application of this method for the analysis of cytosine methylation in low abundance poly(A)RNA using a combination of commercially available kits and standard lab methods to ensure reproducible results. Furthermore, useful information on optimizing the method, suitable controls for almost all steps, and general troubleshooting guides are provided.

Key words

Bisulfite sequencing RNA methylation 5-Methylcytosine 5-Hydroxymethylcytosine mRNA modification 

Notes

Acknowledgment

The research was funded by the Austrian Science Fund (FWF): P27024-BBL.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Division of Molecular Biology, BiocenterMedical University of InnsbruckInnsbruckAustria

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