Improving characterisation of human Multipotent Stromal Cells cultured in 2D and 3D: Design and evaluation of primer sets for accurate gene expression normalisation
Improving characterisation of human Multipotent Stromal Cells cultured in 2D and 3D: Design and evaluation of primer sets for accurate gene expression normalisation
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DOI：
10.1371/journal.pone.0209772
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发表时间：
2018-12-31
期刊：
影响因子：
3.7
通讯作者：
Wang, Hui
中科院分区：
文献类型：
--
作者：
Brinkhof, Bas;Jia, Huidong;Wang, Hui
人多能基质细胞（MSC）是再生医学的宝贵资源，并被广泛研究。它们可以从各种组织中分离出来，并分化成多种细胞类型（多能）。已经发表了许多使用人MSC的报告，并且为了能够比较结果，或者能够识别MSC之间的差异，已经提出了几种细胞表面标记物。然而，仍然存在许多分歧。已知基因表达在细胞阶段和起源之间是不同的。此外，与在支架上生长的细胞（3D）相比，在培养皿上培养的细胞（2D）显示出不同的基因表达谱。甚至RNA提取方法和用于标准化的基因的选择也在基因表达谱中起作用。为了能够比较来自不同维度培养的样品的基因表达数据和使用各种方案提取的RNA，我们着手定义一组适于标准化来自非常异质的样品集的qPCR数据的参考基因。为此，使用Trizol从3D和2D培养的人MSC中提取RNA，以验证新设计和先前公布的引物组。随后，使用RNeasy和Trizol方法从新鲜人MSC样品和储存在RLT缓冲液、Trizol或RNAlater中的样品中提取RNA。在qPCR后，所有样品都已用于根据候选参考基因的稳定性对候选参考基因进行排序，从而能够鉴定最适合用于异质样品集标准化的参考基因。最稳定表达的参照基因表明MSC标志物基因表达上级最不稳定的参照基因的标准化。
Human Multipotent Stromal Cells (MSCs) are a valuable resource for regenerative medicine and are widely studied. They can be isolated from a variety of tissues and differentiate into multiple cell types (multi-potent). Many reports have been published using human MSCs and to be able to compare outcome, or be able to identify differences between MSCs, several cell surface markers have been proposed. Nevertheless, still many differences remain. Gene expression is known to be different between cell stage and origin. Furthermore, cells cultured on a culture dish (2D) show different gene expression profiles as compared to cells grown on scaffolds (3D). Even the RNA extraction method and the selection of genes used for normalisation have a role in gene expression profiling. To be able to compare gene expression data from samples cultured in different dimensions and RNA extracted using a variety of protocols we set out to define a set of reference genes suitable to normalise qPCR data from a very heterogeneous sample set. Hereto, Trizol was used to extract RNA from human MSCs cultured in 3D and 2D to validate newly designed and previously published primer sets. Subsequently, RNA from fresh human MSC samples and samples stored in RLT-buffer, Trizol or RNAlater was extracted using RNeasy and Trizol methods. All samples have been used to rank the candidate reference genes according to their stability after qPCR enabling identification of the most suitable reference gene(s) for normalisation of a heterogeneous sample set. The most stably expressed reference genes indicated superior normalisation of MSC marker gene expression over the least stable reference genes.