A novel RT-QPCR-based assay for the relative quantification of residue specific m6A RNA methylation

Sci Rep. 2019 Mar 12;9(1):4220. doi: 10.1038/s41598-019-40018-6.

Abstract

N6-methyladenosine (m6A) is the most common and abundant RNA modification. Recent studies have shown its importance in the regulation of several biological processes, including the immune response, and different approaches have been developed in order to map and quantify m6A marks. However, site specific detection of m6A methylation has been technically challenging, and existing protocols are long and tedious and often involve next-generation sequencing. Here, we describe a simple RT-QPCR based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues. Using this technique, we have been able to confirm the recently described m6A methylation in the 3'UTR of SOCS1 and SOCS3 transcripts. Moreover, using the method presented here, we have also observed alterations in the relative levels of m6A in specific motifs of SOCS genes in celiac disease patients and in pancreatic β-cells exposed to inflammatory stimuli.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions*
  • Adenosine / analogs & derivatives*
  • Adenosine / genetics
  • Adenosine / metabolism
  • Caco-2 Cells
  • Deoxyribonuclease BamHI / chemistry*
  • Humans
  • Methylation
  • Nucleotide Motifs*
  • Real-Time Polymerase Chain Reaction*
  • Reverse Transcriptase Polymerase Chain Reaction*
  • Suppressor of Cytokine Signaling 1 Protein / genetics
  • Suppressor of Cytokine Signaling 1 Protein / metabolism
  • Suppressor of Cytokine Signaling 3 Protein / genetics
  • Suppressor of Cytokine Signaling 3 Protein / metabolism

Substances

  • 3' Untranslated Regions
  • SOCS1 protein, human
  • SOCS3 protein, human
  • Suppressor of Cytokine Signaling 1 Protein
  • Suppressor of Cytokine Signaling 3 Protein
  • N6-methyladenosine (m6A)
  • Deoxyribonuclease BamHI
  • Adenosine