The emerging field of RNA nanotechnology has led to the utilization of the platform in the field of bionanotechnology as sensing platforms, diverse nanoparticle construction, in vivo computing, and even in vivo drug delivery. Purification of large quantities of supramolecular RNA complexes is of paramount importance due to the large quantities of RNA needed and the purity requirements for in vitro and in vivo assays. Purification is generally carried out by liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), or agarose gel electrophoresis (AGE); however, difficulties exist with each technique. Here, we describe an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradient rate-zonal ultracentrifugation or cushioned sucrose gradient rate-zonal ultracentrifugation. The isolation technique has broad-reaching applicability as production of large quantities of RNA are critical to functional assays involving RNA splicing, RNA secondary structure, anti-sense RNA, and RNA:protein interaction studies.

Learning Objectives:

• Learn about an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradient rate-zonal ultracentrifugation or cushioned sucrose gradient rate-zonal ultracentrifugation
• Learn how this isolation technique has broad-reaching applicability as production of large quantities of RNA are critical to functional assays involving RNA splicing, RNA secondary structure, anti-sense RNA, and RNA:protein interaction studies