Background

RNAscope is a powerful technology that allows for the identification of thousands of RNA targets visualized as puncta, each representing a single RNA transcript, in multiple types of tissues. Use of this assay has steadily increased since its introduction, allowing researchers across biological disciplines to perform rapid and precise quantification of RNA transcripts using a highly sensitive assay. In the neuroscience field, RNAscope is becoming an essential tool. Despite several publications in the field utilizing this technique, there is no standard methodology for processing brain tissue and quantifying RNA transcripts and transcript-positive cells using RNAscope. Many publications provide limited information regarding the calculation of quantification thresholds, preventing replication of prior analyses. Here, we outline a standardized protocol that can be used to establish criteria for quantifying cells that are positive for targets of interest using RNAscope, hopefully allowing for consensus across studies and improving reproducibility across the field. There are many ways in which RNAscope assays and analyses can be conducted; the purpose of this protocol is not to define a correct way, but rather to provide one potential methodology that can be used going forward and, perhaps more importantly, to start a conversation regarding unification and standardization of this approach across neuroscience labs.

Manual quantification of labeled cells is a tedious and time-consuming process. Many available tools used for image analysis (e.g., ImageJ) are often not suitable for analysis of large image files produced in RNAscope studies, and some open-source tools (e.g., dotdotdot or Cell Profiler) require advanced computational skills to run the workflow, thereby limiting their use. Here, we explain how to analyze RNAscope signal in rat neurons using the open-source bioimage quantification software QuPath (Bankhead et al., 2017). This software includes several features such as 1) annotation and visualization tools using a JavaFX interface, 2) built-in algorithms for common tasks, such as cell detection, and 3) interactive machine learning. Our method can be utilized for whole brain quantitative analysis or for analysis in selected brain regions as we performed in Avegno et al. (2021). Although we describe all the steps to generate RNAscope tissue from fresh frozen brains, the same method can be applied to fixed brain preparations with some modifications.

Here, we present the optimization of just one specific parameter important for automated cell detection, the fluorescence intensity threshold. However, we provide the framework for adding optimization steps for additional cell detection parameters, if desired. Collectively, the method presented here offers a user-friendly and cost-effective framework for automated quantification of transcript-positive cells in whole tissue sections, without the need for manual counting.

Materials and Reagents

1. Tissue Preparation

   1. Rodent brains (we used n = 6 adult brains from female Wistar rats purchased from Charles River, catalog number: 003)

   2. Isoflurane, USP, or any other anesthetic (Piramal Critical Care, catalog number: 6679401710; or any other brand)

   3. 250 mL glass beaker (Fisherbrand, catalog number: FB101250; or any brand)

   4. Aluminum foil (Reynold Wraps, catalog number: 458742928317; or any brand)

   5. Ultralow temperature digital thermometer with stainless-steel probe (Fisherbrand, catalog number: 15-077-32)

   6. 2-methylbutane (isopentane) ReagentPlus^® ≥99% (Sigma-Aldrich, catalog number: M32631; or any brand)

   7. Dry ice pellets

   8. Cryostat (Thermo Scientific, USA, Cryostar NX50; or any brand capable of producing 10 µm slices)

   9. Fisherbrand Superfrost Plus microscope slides (Fisher Scientific, catalog number: 12-550-15)

   10. Tissue-Tek O.C.T. compound (Sakura, catalog number: 4583)

   11. Tissue-Tek manual slide staining set (Sakura, catalog number: 4451)

   12. Paraformaldehyde 32% aqueous solution (Electron Microscopy Sciences, catalog number: 15714S)

   13. Distilled water

   14. Fisherbrand Superslip coverslips (Fisher Scientific, catalog number: 12-545-89P)

   15. Fluoro-Gel II with DAPI (Electron Microscopy Sciences, catalog number: 17985-50)

       
       

2. RNAscope Preparation

   1. RNAscope^® Fluorescent Multiplex reagent kit v1 (Advanced Cell Diagnostics, catalog number: 320850) for fresh frozen applications. Note that for fixed brain preparations, additional products are needed: RNAscope Target Retrieval and RNAscope Protease III (available in the RNAscope Universal Pretreatment kit, Advanced Cell Diagnostics, catalog number: 322380)

   2. RNAscope^® RTU Protease IV reagent (Advanced Cell Diagnostics, catalog number: 322340)

   3. RNAscope target probes (catalog number varies depending on the need; we used Rn-Hcrtr1-C1, Rn-Th-C2, and Rn-Fos-C3). Note that in order to independently detect mRNA transcripts in a multiplex assay, each target probe must be in a different channel (C1, C2, or C3) and one of the target probes must be in the C1 channel. Channel C1 target probes are ready-to-use (RTU), while channel C2 and C3 probes are shipped as a 50× concentrated stock. The 50× probes for C2 or C3 must be mixed with a C1 RTU probe. If no specific C1 probe is used, then a blank probe diluent (assay dependent) is used to dilute the probes. Assignation of channels can be changed upon researcher request.

   4. RTU probe diluent (optional, see above; Advanced Cell Diagnostics, USA, catalog number: 300041)

   5. RNAscope 3-plex negative control probes (Advanced Cell Diagnostics, catalog number: 320871)

   6. Immedge^® hydrophobic barrier pen (Vector Labs, catalog number: 310018)

   7. RNase AWAY (Thermo Scientific, catalog number: 14-375-35) or any other decontaminant (e.g., bleach)

      
      

Notes:

1. The items listed below are intended for fresh frozen tissue collection only. If researchers plan to use formalin-fixed tissue, there will be additional steps to be followed (refer to manufacturer manuals for more details).

2. RNAscope kits for fresh frozen and fixed tissues are slightly different, as the fixed tissue follows a different preparation and pretreatment.

3. Target probes listed here are merely indicative. Any type of marker can be utilized for this purpose. Customized probes (from available genome databases) can be made upon request at additional costs.

Equipment

1. Brain extraction tools:

   Straight scissors (Roboz, catalog number: RS-6806), rongeurs (Roboz, catalog number: RS-8300), rounded spatula (Millipore Sigma, catalog number: HS15906), small straight or curved forceps (Roboz, catalog number: RS-5136), and long forceps (Fisherbrand, catalog number: 13-820-077; see Figure 1)

2. Slide scanner (Carl Zeiss, AxioScan Z.1, Germany; or any other microscope capable of scanning and digitizing fluorescent slides)

3. Computer: 64-bit Windows or Linux with minimum 16 GB RAM and a fast multicore processor (e.g., Intel Core i7 or i9)

4. HybEZ II system hybridization oven [Advanced Cell Diagnostics, USA; see Figure 2. The system comprises: HybEZ oven (PN 321710/321720), a humidity control tray (PN 310012), and HybEZ humidifying paper (2 sheets, PN 310025), EZ-Batch wash tray (PN 321717), and EZ-Batch slide holder (PN 321716)]

Note: This protocol will prospectively work with a less powerful computer or with lower RAM, but analysis will be slow and may encounter memory errors.

Figure 1. Brain extraction tools. Straight scissors, rongeurs, rounded spatula, and curved and long forceps indicated.

Figure 2. HybEZ II system. Hybridization oven, humidity control tray, humidifying paper, slide staining/washing set (EZ-Batch wash tray and EZ-Batch slide holder), and hydrophobic pen indicated.

Software

1. Zeiss ZEN blue v2.6 or higher for images acquisition and export (https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html#downloads)

2. QuPath 0.3.2 open-source software (https://qupath.github.io/)

3. Microsoft Excel or similar

4. GraphPad Prism 9.4.1 (https://www.graphpad.com/) or similar

Procedure