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Non-radioactive RNA end-labeling techniques are limited, but more versatile biotin and fluorescent labeling methods are now available. For example, The Thermo Scientific LightShift Chemiluminescent RNA EMSA Kit provides a non-radioactive solution for studying RNA–protein interactions using EMSA. Green stars denote internal IR800CW dye and orange ovals 3 biotin modification. Traditionally, RNA probes are radioactively labeled for detection, although fluorescent and chemiluminescent detection is also possible.

Interestingly, many of the identified RNAs encoded proteins involved in the response to viral infection and RNA metabolism. Specificity is determined through visualization of a single shifted band. To systematically identify the RNAs that interact with NF90, we carried out iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) analysis in the human embryonic fibroblast cell line HEK-293. Alternatively, the protein–RNA complex may be crosslinked and the reaction run on a denaturing gel. Specificity is determined through a competition reaction, where excess unlabeled RNA is incubated in the binding reaction, resulting in a decrease in the shifted signal if the labeled and unlabeled RNA sequences compete for binding of the same protein. This causes a migration shift relative to the unbound RNA probe. Like protein–DNA complexes, a protein–RNA complex migrates more slowly than a free RNA probe through a gel matrix. The binding reaction is then separated via non-denaturing polyacrylamide gel electrophoresis. First, a labeled RNA probe is incubated with a protein sample (typically from a cell lysate) to initiate binding and formation of the interaction complex. Among them, LC-MS/MS and colorimetry can detect the. The current technical methods used to detect m6A include high-throughput sequencing, colorimetry, and LC-MS. The internal modification of mRNA is used to maintain the stability of mRNA.

The RNA electrophoretic mobility shift assay (RNA EMSA) is an in vitro technique used to detect protein–RNA interactions through changes in migration speed during gel electrophoresis. N6-methyladenosine, also called m6A, is a base modification behavior widely found on mRNA.