Analysis and purification of ssRNA and dsRNA molecules using asymmetrical flow field flow fractionation

•AF4 facilitated purification of long ssRNA and dsRNA molecules in native conditions.•AF4 preserved native RNA conformation and enabled detection of RNA multimers.•New information on solution properties of RNA was obtained.•Use of semipreparative AF4 channel allowed milligram RNA loads.•AF4 purified dsRNA molecules triggered RNAi-mediated gene silencing in aphids. Robust RNA purification and analysis methods are required to support the development of RNA vaccines and therapeutics as well as RNA interference -based crop protection solutions. Asymmetrical flow field-flow fractionation (AF4) is a gentle native purification method that applies liquid flows to separate sample components based on their hydrodynamic sizes. We recently showed that AF4 can be utilized to separate RNA molecules that are shorter than 110 nucleotides (nt), but the performance of AF4 in the analysis and purification of longer RNA molecules has not been previously evaluated. Here, we studied the performance of AF4 in separation of single-stranded (ss) and double-stranded (ds) RNA molecules in the size range of 75–6400 nt. In addition, we evaluated the power of AF4 coupling to different detectors, allowing separation to be combined with data collection on yield as well as molecular weight (MW) and size distribution. We show that AF4 method is applicable in RNA purification, quality control, and analytics, and results in good recoveries of ssRNA and dsRNA molecules. In addition, our results demonstrate the utility of AF4 multidetection platforms to study biophysical properties of long RNA molecules.