Simultaneous detection of multiple mRNAs and proteins in bovine IVD cells and tissue with single cell resolution
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ORIGINAL RESEARCH PAPER
Simultaneous detection of multiple mRNAs and proteins in bovine IVD cells and tissue with single cell resolution Kangning Li . Lara Varden . Althea Henderson . Thomas Lufkin Petra Kraus
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Received: 28 June 2020 / Accepted: 1 September 2020 Ó Springer Nature B.V. 2020
Abstract 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.
K. Li L. Varden A. Henderson T. Lufkin P. Kraus (&) Department of Biology, Clarkson University, Potsdam, NY, USA e-mail: [email protected]
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. Keywords Multiprobe RISH/IHC IVD Lamin a/c LMNA YAP/TAZ Ki67 Nucleus pulposus cells
Introduction The intracellular generation of a messenger RNA transcript precedes its translation into a protein. Many diagnostic and research procedures build on detecting RNA transcripts through reverse transcription and polymerase chain reaction (RT-PCR), often in the quantitative real-time manner and primarily on material from pooled cells (Kolodziejczyk et al. 2015; Minogue et al. 2010a; Minogue et al. 2010b; van den Akker et al. 2017; Wang et al. 2020). Cell pooling is a common practice to achieve the critical amount of material for molecular investigation, yet comes at the price of potentially masking cellular dissimilarity within a chosen cell population. Combing methods for single-cell (sc) isolation and culture with advances in scRNA sequencing have pushed the field of sc transcriptomics in recent years. Due to the complex
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nature of the proteome similarly powerful sc proteomics approaches are still undergoing development (Hedlund and Deng 2
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