title: Extraction of high quality RNA from polysaccharide matrices using cetyltrimethylammonium bromide.
abstract: Polysaccharides are increasingly being used as biomaterials for tissue engineering and regenerative medicine. Quantitative analysis of gene expression from cells in three-dimensional (3D) scaffolds requires extraction of messenger RNA, which is complicated in polysaccharide materials by ionic complexing between nucleic acids and the matrix. We used a strongly cationic surfactant, cetyltrimethylammonium bromide (CTAB), to extract RNA from human mesenchymal stem cells embedded in 3D chitosan, agarose and collagen matrices. CTAB extraction was compared to conventional guanidinium thiocyanate-based methods for RNA isolation by assessing RNA yield, purity (A260/A280 and A260/A230) and integrity (28S/18S and RIN). For polysaccharide-based matrices, CTAB extraction yielded significantly more RNA with higher purity than guanidinium thiocyanate-based methods alone. The extracted RNA was largely intact as indicated by 28S/18S ratios and RIN values, while these parameters could not be measured using conventional kits alone. For pure collagen matrices, the CTAB method was comparable or better than guanidinium thiocyanate-based methods in terms of RNA yield and quality. We further validated the CTAB protocol using semi-quantitative and quantitative RT-PCR to amplify both large and small amplicons. Our results show that the CTAB-based method is a facile and effective way to extract abundant, high quality RNA from polysaccharide and protein biomaterials.
authors: Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109, USA.
authors: Limin
authors: L
authors: Wang
authors: Jan P
authors: JP
authors: Stegemann
mesh_terms: D001671:Biochemistry / Q000379:methods*; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000737:chemistry*; D048271:Chitosan / Q000494:pharmacology; D024042:Collagen Type I / Q000494:pharmacology; D005453:Fluorescence; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000187:drug effects / Q000378:metabolism; D011134:Polysaccharides / Q000494:pharmacology*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012685:Sepharose / Q000494:pharmacology
chemical_list: D002593:Cetrimonium Compounds; D024042:Collagen Type I; D011134:Polysaccharides; D012313:RNA; D012685:Sepharose; D048271:Chitosan; D000077286:Cetrimonium