Extraction of high quality RNA from polysaccharide matrices using cetyltrimethylammonium bromide.
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.
Wang
Limin
L
Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109, USA.
Stegemann
Jan P
JP
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
D002593:Cetrimonium Compounds; D024042:Collagen Type I; D011134:Polysaccharides; D012313:RNA; D012685:Sepharose; D048271:Chitosan; D000077286:Cetrimonium