title: Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry.
abstract: RNA interference has provided valuable insight into a wide range of biological systems and is a powerful tool for the analysis of gene function. The exploitation of this pathway to block the expression of specific gene targets holds considerable promise for the development of novel RNAi-based insect management strategies. In addition, there are a wide number of future potential applications of RNAi to control agricultural insect pests as well as its use for prevention of diseases in beneficial insects. The potential to synthesise large quantities of dsRNA by in-vitro transcription or in bacterial systems for RNA interference applications has generated significant demand for the development and application of high throughput analytical tools for the rapid extraction, purification and analysis of dsRNA. Here we have developed analytical methods that enable the rapid purification of dsRNA from associated impurities from bacterial cells in conjunction with downstream analyses. We have optimised TRIzol extractions in conjunction with a single step protocol to remove contaminating DNA and ssRNA, using RNase T1/DNase I digestion under high-salt conditions in combination with solid phase extraction to purify the dsRNA. In addition, we have utilised and developed IP RP HPLC for the rapid, high resolution analysis of the dsRNA. Furthermore, we have optimised base-specific cleavage of dsRNA by RNase A and developed a novel method utilising RNase T1 for RNase mass mapping approaches to further characterise the dsRNA using liquid chromatography interfaced with mass spectrometry.
authors.affiliation: Department of Chemical and Biological Engineering, ChELSI Institute, Mappin Street, University of Sheffield, S1 3JD, UK.
authors.forename: Alison O
authors.initials: AO
authors.lastname: Nwokeoji
authors.affiliation: Department of Chemical and Biological Engineering, ChELSI Institute, Mappin Street, University of Sheffield, S1 3JD, UK.
authors.forename: An-Wen
authors.initials: AW
authors.lastname: Kung
authors.affiliation: Syngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire, RG42 6EY, UK.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Kilby
authors.affiliation: Syngenta, Jealott's Hill International Research Centre, Bracknell, Berkshire, RG42 6EY, UK.
authors.forename: David E
authors.initials: DE
authors.lastname: Portwood
authors.affiliation: Department of Chemical and Biological Engineering, ChELSI Institute, Mappin Street, University of Sheffield, S1 3JD, UK. Electronic address: m.dickman@sheffield.ac.uk.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Dickman
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D007313:Insecta; D013058:Mass Spectrometry; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D012259:Ribonuclease, Pancreatic; D017423:Sequence Analysis, RNA
chemical_list: D012330:RNA, Double-Stranded; D012259:Ribonuclease, Pancreatic
keywords: Double stranded RNA purification; Ion pair reverse phase HPLC; Mass spectrometry; RNase mass mapping