As with other procedures predicated on biochemical alteration followed by sequencing, the development of high-throughput sequencing has removed the limitations requiring prior knowledge of sites of interest and primer design. The method causes a lot of RNA degradation, so it is necessary to start with a large amount of sample, or use effective normalisation techniques to account for amplification biases. One final limitation is that, for CMC labelling of pseudouridine to be specific, it is not complete, and therefore nor is it quantitative. A new reactant that could achieve a higher sensitivity with specificity would be beneficial.