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

Mohamad Alameh, Myriam Jean, Diogo DeJesus, Michael D Buschmann, Abderrazzak Merzouki1Institute of Biomedical Engineering, Department of Chemical Engineering, &Eacute;cole Polytechnique, Station Centre-ville, Montr&eacute;al, QC, CanadaAbstract: Chitosan, a well known natural cationi...

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Autores principales:	Mohamad Alameh, Myriam Jean, Diogo DeJesus, et al
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2010
Materias:	Medicine (General) R5-920
Acceso en línea:	https://doaj.org/article/3573f281e2644621ab9b06f1c222391f
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spelling	oai:doaj.org-article:3573f281e2644621ab9b06f1c222391f2021-12-02T01:35:40ZChitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing1176-91141178-2013https://doaj.org/article/3573f281e2644621ab9b06f1c222391f2010-07-01T00:00:00Zhttp://www.dovepress.com/chitosanase-based-method-for-rna-isolation-from-cells-transfected-with-a4776https://doaj.org/toc/1176-9114https://doaj.org/toc/1178-2013Mohamad Alameh, Myriam Jean, Diogo DeJesus, Michael D Buschmann, Abderrazzak Merzouki1Institute of Biomedical Engineering, Department of Chemical Engineering, &Eacute;cole Polytechnique, Station Centre-ville, Montr&eacute;al, QC, CanadaAbstract: Chitosan, a well known natural cationic polysaccharide, has been successfully &shy;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 Mohamad AlamehMyriam JeanDiogo DeJesuset alDove Medical PressarticleMedicine (General)R5-920ENInternational Journal of Nanomedicine, Vol 2010, Iss default, Pp 473-481 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine (General) R5-920
spellingShingle	Medicine (General) R5-920 Mohamad Alameh Myriam Jean Diogo DeJesus et al Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing
description	Mohamad Alameh, Myriam Jean, Diogo DeJesus, Michael D Buschmann, Abderrazzak Merzouki1Institute of Biomedical Engineering, Department of Chemical Engineering, &Eacute;cole Polytechnique, Station Centre-ville, Montr&eacute;al, QC, CanadaAbstract: Chitosan, a well known natural cationic polysaccharide, has been successfully &shy;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
format	article
author	Mohamad Alameh Myriam Jean Diogo DeJesus et al
author_facet	Mohamad Alameh Myriam Jean Diogo DeJesus et al
author_sort	Mohamad Alameh
title	Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing
title_short	Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing
title_full	Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing
title_fullStr	Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing
title_full_unstemmed	Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing
title_sort	chitosanase-based method for rna isolation from cells transfected with chitosan/sirna nanocomplexes for real-time rt-pcr in gene silencing
publisher	Dove Medical Press
publishDate	2010
url	https://doaj.org/article/3573f281e2644621ab9b06f1c222391f
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Chitosanase-based method for RNA isolation from cells transfected with chitosan/siRNA nanocomplexes for real-time RT-PCR in gene silencing

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