SCARLET (site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography) is used determining the fraction of RNA in a sample that carries a methylated adenine at a specific site. One can start with total RNA without having to enrich for the target RNA molecule. Therefore, it is an especially suitable method for quantifying methylation status in low abundance RNAs such as tRNAs. However, it is not suitable or practical for large-scale location of m6A sites.
The procedure begins with a chimeric DNA oligonucleotide annealing to the target RNA around the candidate modification site. The chimeric ssDNA has 2’OMe/2’H modifications and is complementary to the target sequence. The chimeric oligonucleotide serves as a guide to allow RNase H to cleave the RNA strand precisely at the 5’-end of the candidate site. The cut site is then radiolabeled with phosphorus-32 and splint-ligated to a 116nt ssDNA oligonucleotide using DNA ligase. RNase T1/A is introduced to the sample to digest all RNA, except for the RNA molecules with the 116-mers DNA attached. This radiolabeled product is then isolated and digested by nuclease to generate a mixture of modified and unmodified adenosines (5’P-m6A and 5’-P-A) which is separated using thin layer chromatography. The relative proportions of the two groups can be determined using UV absorption levels.