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.

Keywords: IVD; Ki67; LMNA; Lamin a/c; Multiprobe RISH/IHC; Nucleus pulposus cells; YAP/TAZ.

Fig. 1. Illustration of RISH/IHC procedures.

Simplified sequential procedure for single or double RISH with IHC (a) or RISH with double IHC (b) alongside an approximate timeline for the individual modules of the procedure. A schematic of a cell illustrates the different components and their targets after double RISH and IHC (c) or RISH and double IHC (d). 488,568,594 and 647 indicate the different Alexa-fluorophore conjugated antibodies; t1/t2: different RNA transcripts; px/py: different target proteins; mad: mouse anti-DIG antibody; maf: mouse anti-FITC antibody; gam: goat anti-mouse antibody; rax: rabbit-anti protein x antibody; ray: rabbit anti-protein y antibody: gar: goat ant-rabbit antibody; W/B: wash and blocking; W: wash.

Fig. 2. Example of the double RISH procedure for transcripts of the mechanotransduction components YAP, TAZ and LMNA on sections through the coccygeal bovine IVD.

(a-d) shows cells of the AF. (e-h) shows cells of the NP. Alexa-488 conveyed fluorescence reflects a RNA probe targeting exon2 of LMNA in (a,c,e and g) or the TAZ transcript in (b,d,f and h). Alexa-594 conveyed fluorescence indicates the presence of a RNA probe targeting exon 13 of LMNA in (a,d,e and h) or the YAP transcript in (b,c,f and g). The white arrowhead in (h) points to a potential progenitor cell as indicated by the absence LMNA or TAZ transcripts. DAPI stains the nucleus

Fig. 3. Example of a combined RISH and IHC procedure for cell cycle markers Ki67 and CDK4 on primary bovine NP cells in culture.

Alexa-488 conveyed fluorescence reflects the bovine Ki67 transcript, Alexa-568 conveyed fluorescence reflects the presence of Ki67 protein, while Alexa-647 conveyed fluorescence indicates presence of CDK4 protein. DAPI stains the nucleus. The white arrowhead in (e) points to a NP cell in metaphase. The green and red arrowheads in (f-j) point to Ki67 transcript and Ki67 protein, respectively