Effect of N1-methyladenosine in the quantification of RNA

Abstract The reverse transcription (RT) of RNA to cDNA is a key step for the quantification of nucleic acid molecules in numerous basic research and medical diagnosis. Although multiple sources of errors have been considered, little is known about the impact of RNA modifications on the validity of genes of interest for quantitative RT-PCR. Here, we evaluated the influence of RNA modifications of N1-methyladenosine (m1A) on the validity of the RT step by quantifying two RNAs with commercial reverse transcriptase and RNA sample from HEK-293T cells or in vitro transcription. Our findings prove that RNA modification of m1A is a source of RT variability as it acts as an arrest signal of RT at its position, in turn affecting the corresponding RNA quantification.