Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing

Abstract

Mohamad Alameh, Myriam Jean, Diogo DeJesus, Michael D Buschmann, Abderrazzak Merzouki1Institute of Biomedical Engineering, Department of Chemical Engineering, École Polytechnique, Station Centre-ville, Montréal, QC, CanadaAbstract: Chitosan, a well known natural cationic polysaccharide, has been successfully ­implemented in vitro and in vivo as a nonviral delivery system for both plasmid DNA and siRNA. While using chitosan/siRNA polyplexes to knock down specific targets, we have underestimated the effect of nucleic acids binding to chitosan when extracting RNA for subsequent quantitative PCR evaluation of silencing. In vitro transfection using chitosan/siRNA-based polyplexes reveals a very poor recovery of total RNA especially when using low cell numbers in 96 well plates. Here, we describe a method that dramatically enhances RNA extraction from chitosan/siRNA-treated cells by using an enzymatic treatment with a type III chitosanase. We show that chitosanase treatment prior to RNA extraction greatly enhances the yield and the integrity of extracted RNA. This method will therefore eliminate the bias associated with lower RNA yield and integrity when quantifying gene silencing of chitosan-based systems using quantitative real time PCR.Keywords: chitosan, chitosanase, siRNA, DPP-IV gene silencing, RIN, qPCR