Spatial High-resolution Analysis of Gene Expression Levels in Tendons
Summary
This article describes how to perform an optimized in situ protocol for tendons. This method discusses tissue preparation, section permeabilization, probe design, and signal amplification methods.
Abstract
In recent years, many protocols have been developed for high-resolution transcriptomics in many different medical and biology fields. However, matrix-rich tissues, and specifically, tendons were left behind due to their low cell number, low RNA amount per cell, and high matrix content, which made them complicated to analyze. One of the recent and most important single-cell tools is the spatial analysis of gene expression levels in tendons. These RNA spatial tools have specifically high importance in tendons to locate specific cells of new and unknown populations, validate single-cell RNA-seq results, and add histological context to the single-cell RNA-seq data. These new methods will enable the analysis of RNA in cells with exceptional sensitivity and the detection of single-molecule RNA targets at the single-cell level, which will help to molecularly characterize tendons and promote tendon research.
In this method paper, we will focus on the available methods to analyze spatial gene expression levels on histological sections by using novel in situ hybridization assays to detect target RNA within intact cells at single-cell levels. First, we will focus on how to prepare the tendon tissue for the different available assays and how to amplify target-specific signals without background noise but with high sensitivity and high specificity. Then, the paper will describe specific permeabilization methods, the different probe designs, and the signal amplification strategies currently available. These unique methods of analyzing transcription levels of different genes in single-cell resolution will enable the identification and characterization of the tendon tissue cells in young and aged populations of various animal models and human tendon tissues. This method will also help analyze gene expression levels in other matrix-rich tissues such as bones, cartilage, and ligaments.
Introduction
Tendons are connective tissues that enable the transmission of force between muscle and bone1. Developmentally, axial tenocytes are derived from mesenchymal cells within the sclerotome of the somites2; limb tendons derive from the lateral plate mesoderm; and cranial tendons arise from the cranial neural crest lineage3,4. Tendon can be characterized by the expression of the scleraxis transcription factor5, although several markers also play a key role in tendon development, including tenomodulin, mohawk, and early growth response 1/26,7,8,9.
Despite the few known markers of the tendon, in general, a more in-depth characterization remains challenging because the tendon contains cells that span across a gradient of biomechanical properties. From the myotendinous junction, tendon mid-body, and the more calcified enthesis, the tendon cells reside in extracellular matrices that range in tensile properties. Since the tendon must withstand tensile stress imposed by the difference in mechanical strength between soft and hard tissue, the spatial organization of the cells in the tendon is particularly important for its function. However, little is known about these tendon subpopulations.
Many high-resolution spatial transcriptomic tools can be used to begin to elucidate cell subpopulations, including but not limited to, single-cell RNA Seq or in situ hybridization. However, while these spatial profiling assays help uncover RNA expression across tissue after microdissection or sectioning, these methods can be challenging when performed on tendon tissue. Tendons are matrix-rich tissues composed of nearly 86% of collagen by dry mass10, making it challenging to extract the cells for sequencing. Due to both the complications in isolating cells from the matrix, the hypocellular nature of the tendon11, and the relatively low RNA count, the tendon is a difficult tissue to analyze.
In this paper, we present a method to optimize novel in situ hybridization assays to leverage them for tendons by providing tissue preparation, permeabilization, and probe design methods. Coupled with existing sequencing technologies, this may help researchers spatially characterize tendon subpopulations across developing, adult, or injured tendons with increased assay sensitivity and specificity.
Protocol
Representative Results
Discussion
In this paper, we describe modifications made to leverage existing ISH tools such that they can be used in tendon tissue with a high degree of specificity and sensitivity. Since the tendon is a highly matrix-dense tissue, protocol adjustments must often be made to achieve similar degrees of probe penetration and specificity. These specific permeabilization methods and signal amplification strategies of the tendon tissue are integral to improving the efficacy of the ISH protocols discussed. Without these steps, it is chal…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Jenna Galloway and the members of Galloway Lab for their support and encouragement in the development and troubleshooting of these protocols.
Materials
| 1 M triethanolamine buffer | |||
| 10% Formalin solution | |||
| 10% Tween-20 | |||
| 20x Saline Sodium Citrate buffer | |||
| 4% PFA | |||
| ACD RNAscope Fluorescent Multiplex Fluorescent Reagent Kit V2 | ACD | 323100 | |
| Acetic Anhydride | |||
| Axio Imager Microscope | ZEISS | ||
| C57BL/J6 mice | JAX ID: 000664 | ||
| Coverslips | Fisher | 12-541-042 | |
| ddH2O | |||
| ETDA | Thermofisher | AM9262 | |
| EtOH | |||
| Glucose | VWR Chemicals BDH | BDH9230-500G | |
| HCR RNA-FISH Bundle | Molecular Instruments Inc. | ||
| HybEZ II Hybridization System | ACD | ||
| Immedge Barrier Pen | Vector Laboratories | H4000 | |
| Leica SPE Confocal Microscope | Leica | ||
| Parafilm | Fisher | ||
| Phosphate-buffered saline (PBS, 1x) | Invitrogen | AM9625 | Dilute 10x PBS in milli-Q water to get 1x solution |
| Protease IV | |||
| Proteinase K | Roche | 3115836001 | |
| RNAscope H2O2 and Protease Reagents | ACD | PN 322381 | Included in ACD RNAscope Fluorescent Multiplex Fluorescent Reagent Kit V3 |
| RNAscope Multiplex Fluorescent Detection Kit | ACD | PN 323110 | Included in ACD RNAscope Fluorescent Multiplex Fluorescent Reagent Kit V2 |
| RNAscope Target Retrieval reagents | ACD | 322000 | Included in ACD RNAscope Fluorescent Multiplex Fluorescent Reagent Kit V4 |
| RNAscope Wash Buffer | ACD | PN 310091 | Included in ACD RNAscope Fluorescent Multiplex Fluorescent Reagent Kit V5 |
| RNAscope Probe Diluent | ACD | 300041 | |
| Slide holder | StatLab | 4465A | |
| Staining Dish with Lid | StatLab | LWS20WH | |
| Superfrost Plus Microscope slides | Fisher | 1255015 | treated, charged slides |
| Tris-HCl | |||
| Xylene | Sigma-Aldrich | 534056-4L |
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