Department of Biomedical Engineering, University of Michigan, 1101 Beal Ave., Ann Arbor, MI 48109, USA.
Biomaterials. 2010 Mar;31(7):1612-8. doi: 10.1016/j.biomaterials.2009.11.024. Epub 2009 Dec 3.
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.
多糖越来越多地被用作组织工程和再生医学的生物材料。从三维（3D）支架中的细胞定量分析基因表达需要提取信使 RNA，但在多糖材料中，核酸与基质之间的离子络合会使 RNA 的提取变得复杂。我们使用强阳离子表面活性剂十六烷基三甲基溴化铵（CTAB）从嵌入 3D 壳聚糖、琼脂糖和胶原蛋白基质中的人间充质干细胞中提取 RNA。通过评估 RNA 产量、纯度（A260/A280 和 A260/A230）和完整性（28S/18S 和 RIN），将 CTAB 提取与传统的基于胍硫氰酸盐的 RNA 分离方法进行比较。对于基于多糖的基质，CTAB 提取的 RNA 产量明显更高，纯度也更高，优于单独使用基于胍硫氰酸盐的方法。提取的 RNA 基本完整，28S/18S 比值和 RIN 值表明了这一点，而单独使用传统试剂盒则无法测量这些参数。对于纯胶原蛋白基质，CTAB 方法在 RNA 产量和质量方面与基于胍硫氰酸盐的方法相当或更好。我们进一步使用半定量和定量 RT-PCR 验证了 CTAB 方案，以扩增大、小扩增子。我们的结果表明，CTAB 法是一种从多糖和蛋白质生物材料中提取丰富、高质量 RNA 的简便有效方法。
