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.
journal_name
Biomaterialsjournal_title
Biomaterialsauthors
Wang L,Stegemann JPdoi
10.1016/j.biomaterials.2009.11.024subject
Has Abstractpub_date
2010-03-01 00:00:00pages
1612-8issue
7eissn
0142-9612issn
1878-5905pii
S0142-9612(09)01252-6journal_volume
31pub_type
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