Single-Step RNA Extraction from Different Hydrogel-Embedded Mesenchymal Stem Cells for Quantitative Reverse Transcription-Polymerase Chain Reaction Analysis
Single-Step RNA Extraction from Different Hydrogel-Embedded Mesenchymal Stem Cells for Quantitative Reverse Transcription-Polymerase Chain Reaction Analysis
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DOI：
10.1089/ten.tec.2015.0362
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发表时间：
2016-06-01
影响因子：
3
通讯作者：
Salzig, Denise
中科院分区：
文献类型：
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作者：
Koester, Natascha;Schmiermund, Alexandra;Salzig, Denise
对于许多组织工程应用，细胞如人间充质干细胞（hMSC）必须包埋在水凝胶中。包埋的hMSC的分析需要RNA提取，但常见的提取程序往往产生低产量和/或质量差的RNA。我们系统地研究了四种均质化方法结合八种RNA提取方案，用于包埋在三种常见水凝胶类型（藻酸盐，琼脂糖和明胶）中的hMSCs。我们发现对于所有三种水凝胶类型，使用液氮或转子-定子产生低RNA产率，而使用微匀浆器或酶/化学水凝胶消化实现更好的产率，无论随后应用哪种提取方案。热苯酚提取方案通常获得最高的A（260）值（代表每10（6）个细胞高达40.8 μ g RNA），但十六烷基三甲基溴化铵（CTAB）方法产生的RNA质量更好，具有A（260）/A（280）和A（260）/A（230）比率和紫外光谱与纯RNA对照相似。通过该方法产生的RNA也适合作为终点和定量逆转录-PCR（qRT-PCR）的模板，实现了类似于20的低C-t值。谨慎选择水凝胶匀浆和RNA提取方法可以确保制备高质量的RNA，从而产生可靠的终点和定量RT-PCR数据。因此，我们提出了一种通用的方法，适用于提取RNA的细胞包埋在所有三种水凝胶类型通常用于组织工程。
For many tissue engineering applications, cells such as human mesenchymal stem cells (hMSCs) must be embedded in hydrogels. The analysis of embedded hMSCs requires RNA extraction, but common extraction procedures often produce low yields and/or poor quality RNA. We systematically investigated four homogenization methods combined with eight RNA extraction protocols for hMSCs embedded in three common hydrogel types (alginate, agarose, and gelatin). We found for all three hydrogel types that using liquid nitrogen or a rotor-stator produced low RNA yields, whereas using a microhomogenizer or enzymatic/chemical hydrogel digestion achieved better yields regardless of which extraction protocol was subsequently applied. The hot phenol extraction protocol generally achieved the highest A(260) values (representing up to 40.8 mu g RNA per 10(6) cells), but the cetyltrimethylammonium bromide (CTAB) method produced RNA of better quality, with A(260)/A(280) and A(260)/A(230) ratios and UV spectra similar to the pure RNA control. The RNA produced by this method was also suitable as a template for endpoint and quantitative reverse transcription-PCR (qRT-PCR), achieving low C-t values of similar to 20. The prudent choice of hydrogel homogenization and RNA extraction methods can ensure the preparation of high-quality RNA that generates reliable endpoint and quantitative RT-PCR data. We therefore propose a universal method that is suitable for the extraction of RNA from cells embedded in all three hydrogel types commonly used for tissue engineering.