Dataset Information

Simultaneous detection of multiple mRNAs and proteins in bovine IVD cells and tissue with single cell resolution.

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.

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.

SUBMITTER: Li K

PROVIDER: S-EPMC7796921 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundWhole-mount in situ hybridization (WISH) is a fundamental tool for studying the spatio-temporal expression pattern of RNA molecules in intact embryos and tissues. The available methodologies for detecting mRNAs in embryos rely on enzymatic activities and chemical reactions that generate diffusible products, which are not fixed to the detected RNA, thereby reducing the spatial resolution of the technique. In addition, current WISH techniques are time-consuming and are usually not combined with methods reporting the expression of protein molecules.ResultsThe protocol we have developed and present here is based on the RNAscope technology that is currently employed on formalin-fixed, paraffin-embedded and frozen tissue sections for research and clinical applications. By using zebrafish embryos as an example, we provide a robust and rapid method that allows the simultaneous visualization of multiple transcripts, demonstrated here for three different RNA molecules. The optimized procedure allows the preservation of embryo integrity, while exhibiting excellent signal-to-noise ratios. Employing this method thus allows the determination of the spatial expression pattern and subcellular localization of multiple RNA molecules relative to each other at high resolution, in the three-dimensional context of the developing embryo or tissue under investigation. Lastly, we show that this method preserves the function of fluorescent proteins that are expressed in specific cells or cellular organelles and conserves antigenicity, allowing protein detection using antibodies.ConclusionsBy fine-tuning the RNAscope technology, we have successfully redesigned the protocol to be compatible with whole-mount embryo samples. Using this robust method for zebrafish and extending it to other organisms would have a strong impact on research in developmental, molecular and cell biology. Of similar significance would be the adaptation of the method to whole-mount clinical samples. Such a protocol would contribute to biomedical research and clinical diagnostics by providing information regarding the three-dimensional expression pattern of clinical markers.