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

• Published in: International journal of nanomedicine Vol. 5; no. default; pp. 473 - 481
• Main Authors: Alameh, Mohamad, Jean, Myriam, Dejesus, Diogo, Buschmann, Michael D, Merzouki, Abderrazzak
• Format: Journal Article
• Language: English
• Published: New Zealand Taylor & Francis Ltd 01-01-2010; Dove Press; Dove Medical Press
• Subjects: Caco-2 Cells; chitosan; Chitosan - chemistry; chitosanase; DPP-IV gene silencing; Drug Delivery Systems; Gene Silencing; Glycoside Hydrolases; Hep G2 Cells; HT29 Cells; Humans; In Vitro Techniques; Nanomedicine; Nanoparticles - administration & dosage; Nanoparticles - chemistry; Original Research; qPCR; Reverse Transcriptase Polymerase Chain Reaction; RIN; RNA - genetics; RNA - isolation & purification; RNA, Small Interfering - administration & dosage; RNA, Small Interfering - genetics; siRNA; Transfection; chitosan; qPCR; siRNA; DPP-IV gene silencing; RIN; chitosanase
• ISSN: 1178-2013; 1176-9114; 1178-2013
• DOI: 10.2147/ijn.s10879

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.