Abstract
The use of RNA electrophoretic mobility shift assays (REMSAs) for analysis of RNA–protein interactions have been limited to lengthy assay time and qualitative assessment. To vastly improve assay efficiency, feasibility and quality of data procured from REMSAs, we combine here some of the best-known labeling and electrophoretic techniques. Nucleic acid fragments are end-labeled with fluorescent tags, as opposed to the radioactive or biotin tags. The fluorescent probes may be detected directly from the electrophoresis gel, eliminating the need for cumbersome membrane transfer and immunoblotting. Modifying the REMSA protocol to include low-molarity, lithium borate conductive media and near-infrared-labeled probes allows for a reduction assay time, quantitative comparison between experimental conditions and crisp band resolution (i.e., optimized results).
METHOD SUMMARY
We present an improvement in resolution, speed and ease of RNA electrophoretic mobility shift assays. First, sensitive and quantitative detection of gel shifts can be improved with near-infrared tagged RNA oligos, as opposed to more toxic and cumbersome labeling methods, such as radioisotopes or biotin tags. Second, for improvement of resolution and efficiency, traditional Tris-based conductive running media are replaced with low-molarity, lithium borate-conductive media. These improvements to the methodology significantly reduce assay time, as well as improve the quality and overall utility of this technique in the study of RNA–protein interactions in vitro.
Papers of special note have been highlighted as: • of interest; •• of considerable interest
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