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Simultaneous detection of multiple mRNAs and proteins in bovine IVD cells and tissue with single cell resolution
Biotechnology Letters ( IF 2.0 ) Pub Date : 2020-09-09 , DOI: 10.1007/s10529-020-02997-9
Kangning Li 1 , Lara Varden 1 , Althea Henderson 1 , Thomas Lufkin 1 , Petra Kraus 1
Affiliation  

Objectives Interactions of cells with their neighbors and influences by the surrounding extracellular matrix (ECM) is reflected in a cells transcriptome and proteome. In tissues comprised of heterogeneous cell populations or cells depending on ECM signalling cues such as those of the intervertebral disc (IVD), this information is obscured or lost when cells are pooled for the commonly used transcript analysis by quantitative PCR or RNA sequencing. Instead, these cells require means to analyse RNA transcript and protein distribution at a single cell or subcellular level to identify different cell types and functions, without removing them from their surrounding signalling cues. Results We developed a simple, sequential protocol combining RNA is situ hybridisation (RISH) and immunohistochemistry (IHC) for the simultaneous analysis of multiple transcripts alongside proteins. This allows one to characterize heterogeneous cell populations at the single cell level in the natural cell environment and signalling context, both in vivo and in vitro. This protocol is demonstrated on cells of the bovine IVD, for transcripts and proteins involved in mechanotransduction, stemness and cell proliferation. Conclusions A simple, sequential protocol combining RISH and IHC is presented that allows for simultaneous information on RNA transcripts and proteins to characterize cells within a heterogeneous cell population and complex signalling environments such as those of the IVD.

中文翻译:

以单细胞分辨率同时检测牛体外诊断细胞和组织中的多种 mRNA 和蛋白质

目的细胞与其邻居的相互作用以及周围细胞外基质 (ECM) 的影响反映在细胞转录组和蛋白质组中。在由异质细胞群或细胞组成的组织中,取决于 ECM 信号提示,例如椎间盘 (IVD),当细胞被汇集用于通过定量 PCR 或 RNA 测序进行常用的转录分析时,这些信息会被掩盖或丢失。相反,这些细胞需要在单个细胞或亚细胞水平上分析 RNA 转录物和蛋白质分布的方法,以识别不同的细胞类型和功能,而无需将它们从周围的信号线索中去除。结果我们开发了一个简单的,结合 RNA 的顺序协议是原位杂交 (RISH) 和免疫组织化学 (IHC),用于同时分析多个转录物和蛋白质。这允许人们在自然细胞环境和信号环境中,在体内和体外在单细胞水平上表征异质细胞群。该协议在牛 IVD 细胞上得到证明,用于参与机械转导、干性和细胞增殖的转录物和蛋白质。结论 提出了一种结合 RISH 和 IHC 的简单、顺序协议,它允许同时提供有关 RNA 转录物和蛋白质的信息,以表征异质细胞群和复杂信号环境(如 IVD 的信号环境)中的细胞。这允许人们在自然细胞环境和信号环境中,在体内和体外在单细胞水平上表征异质细胞群。该协议在牛 IVD 细胞上得到证明,用于参与机械转导、干性和细胞增殖的转录物和蛋白质。结论 提出了一种结合 RISH 和 IHC 的简单、顺序协议,它允许同时提供有关 RNA 转录物和蛋白质的信息,以表征异质细胞群和复杂信号环境(如 IVD 的信号环境)中的细胞。这允许人们在自然细胞环境和信号环境中,在体内和体外在单细胞水平上表征异质细胞群。该协议在牛 IVD 细胞上得到证明,用于参与机械转导、干性和细胞增殖的转录物和蛋白质。结论 提出了一种结合 RISH 和 IHC 的简单、顺序协议,它允许同时提供有关 RNA 转录物和蛋白质的信息,以表征异质细胞群和复杂信号环境(如 IVD 的信号环境)中的细胞。
更新日期:2020-09-09
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