Site-specific analysis of ribosomal 2′O-methylation by quantitative reverse transcription PCR under low deoxynucleotide triphosphate concentrations
Daniela Barros-Silva,
Johan Tsui,
Carmen Jerónimo,
Guido Jenster,
Elena S Martens-Uzunova
Affiliations
Daniela Barros-Silva
1Department of Urology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Be-331, PO Box 2040, 3000 CA Rotterdam, The Netherlands
Johan Tsui
1Department of Urology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Be-331, PO Box 2040, 3000 CA Rotterdam, The Netherlands
Carmen Jerónimo
2Cancer Biology & Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Centre (Porto CCC), Rua Dr. António Bernardino de Almeida, Porto, 4200-072, Portugal
Guido Jenster
1Department of Urology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Be-331, PO Box 2040, 3000 CA Rotterdam, The Netherlands
Elena S Martens-Uzunova
1Department of Urology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Be-331, PO Box 2040, 3000 CA Rotterdam, The Netherlands
Ribose 2′O-methylation (Nm, ribomethylation) is the most abundant RNA modification present in rRNA. It has been shown that alterations in ribosomal 2′O-methylation at individual Nm sites likely reflect regulated cellular processes. Although several analytical approaches for Nm detection and profiling have been developed, a simple and affordable method for the screening and measurement of individual Nm sites in large numbers of tissue samples is required to examine their potential for clinical translation. Here, we describe a new quantitative reverse transcription PCR-based method that can sensitively assess ribomethylation levels at specific rRNA sites at single-nucleotide resolution in low input amounts of total RNA.