Fast Analysis of RNA from Transcription and Rho-Dependent Termination Assays Using Automated Capillary...

Conventional analysis of RNA products synthesized by bacterial RNA polymerase (RNAP) in in vitro transcription assays relies on radioactive labeling, followed by separation of labeled RNAs by denaturing urea-polyacrylamide gel electrophoresis (PAGE). This approach provides excellent resolution across a wide range of RNA lengths (from 2 nt to several hundred nucleotides), but the use of radioisotopes requires specialized facilities, strict waste-disposal procedures, and is often restricted. To circumvent these limitations, we adapted a standard in vitro transcription assay for use with the Agilent 2100 Bioanalyzer capillary electrophoresis system. Here, we describe the application of this method to analyze run-off transcription by Mycobacterium tuberculosis (Mtb) RNAP and its transcription termination products generated by the Mtb Rho factor. We demonstrate that RNAs produced in in vitro transcription reactions can be detected and quantified without radioactive labeling, simplifying and accelerating the workflow. Although capillary electrophoresis lacks the single-nucleotide resolution of denaturing urea-PAGE, it provides a practical alternative that is compatible with any laboratory environment. This protocol is broadly applicable for analyzing RNA products generated by in vitro transcription with any bacterial RNAP, in the presence or absence of RNA remodeling factors such as the Rho protein.