Table 1.

Summary of techniques for the detection of RNA cytosine-5 methylation (5-mrC)

Level	Techniques	Principle	Advantages	Disadvantages	Resolution	Data analysis
Global level	RNA dot blot	antibody-antigen reaction	Quick to obtain result	Qualitative or semi-quantitative data	Relative global 5-mrC level	t-test
	ELISA-based 5-mrC detection	antibody-antigen reaction	Quantitative measurement of 5-mrC level; commercially-available kit	Unable to detect locus-specific 5-mrC level	Absolute global 5-mrC abundance	t-test
	LC-MS/MS	mass spectrometry	Accurate quantitative measurement of 5-mrC abundance	Unable to detect locus-specific 5-mrC level	Absolute global 5-mrC abundance	t-test
Transcriptome-wide level	5-mrC-RIP-seq	RNA immunoprecipitation	Enable to enrich low abundance RNAs with 5-mrC modification	IP induces background noise; not single nucleotide resolution	100nt~200nt resolution	MACS for peak calling; compare peak intensity between groups
	Aza-IP-seq	Protein immunoprecipitation	Enable to identify specific enzyme target 5-mrC sites	IP induces background noise; 5-Azais toxic to cells	enzyme-specific nucleotide resolution	meRanTK: mapping, methylation calling (C to G transversion), enrichment comparison
	miCLIP-seq	Protein immunoprecipitation	Enable to identify specific enzymetarget 5-mrC sites	The generation of mutant enzymes is time-consuming and costy	enzyme-specific nucleotide resolution	mapping, methylation calling (end of sequencing reads)
	RNA Bisulfite sequencing	Bisulfite conversion of unmethylated cytosine to uracil while methylated cytosine remains unchanged	Provide unbiased transcriptome-wide single nucleotide view	Difficult to detect 5-mrC modification in low abundance RNAs due to degradation during bisulfite conversion process	single-nucleotide resolution	Bioinformatic tools: meRanTK, BS-RNA; BisRNA
Locus-specific level	5-mrC-RIP followed by RT-qPCR	RNA immunoprecipitation	Enable to measure the relative methylation level of specific 5-mrC sites.	IP induces background noise	Locus-specific level	t-test
	RNA bisulfite conversion combined with PCR amplicon-based or cloning-based Sanger sequencing	Sanger sequencing	Enable to detect the methylation level of specific 5-mrC sites	sequencing depth	Locus-specific level	t-test
	RNA bisulfite PCR combined with pyrosequencing	pyrosequencing	Enable to detect the methylation level of specific 5-mrC sites	Primer design is sequence context-dependent.	Locus-specific level	t-test

Abbreviations: IP: immunoprecipitation