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Are You In(to) Situ? – Putting Together Your First RNAscope® Assay

Posted in: DNA / RNA Manipulation and Analysis

Content sponsored by New England Biolabs

RNA in situ hybridization - Human Melanoma FFPE Tissue Section (KRT5 and Housekeeping Gene)

You are thinking of trying out RNAscope®. After all, RNAscope® holds promise for increasing the sensitivity and specificity of your in situ hybridization. Yet, getting started can be a little overwhelming with the numerous kits and reagents available in the RNAscope product line. Here’s an overview of your options to help you navigate to the right setup for you!

The Whole Kit and Kaboodle – Selecting a Kit

The first thing you’ll have to consider when selecting a kit is whether you want to stain for a single or for multiple RNA sequences of interest at one time. If you want to detect three (sometimes four) RNA sequences simultaneously in the same tissue (e.g. for colocalization analysis), you’ll want to select a kit that can use one color per target. Kits that use a different color “channel” for each distinct signal are called multiplex kits.

You also have the option of selecting either a chromogenic or fluorescent kit. Chromogenic assays can be used to multiplex up to two distinct targets (duplex), are visualized easily under a simple brightfield microscope, and result in permanent stains.  Fluorescent assays allow for multiplexing up to three distinct targets, but the signal is light sensitive and degrades from looking at it under the microscope for extended periods of time. In some cases, up to four targets can be labelled, but this is only recommended for certain tissue types and requires additional considerations.

Each chromogenic kit has different color options. The RNAscope® 2.5 HD Brown kit will make your RNA molecules look like little brown specks. The RNAscope® 2.5 HD Red kit will make them red specks. Then the duplex RNAscope® 2.5 HD Brown/Red will allow you to stain one target red while the other brown. Pretty straight forward.

The fluorescent kits only come in two flavors – RNAscope® Fluorescent Multiplex and Multiplex Fluorescent v2. The kits are universally compatible with different color combinations, allowing for maximal flexibility with one to three targets. The RNAscope® Multiplex Fluorescent v2 kit is usually only recommended for formalin-fixed paraffin embedded (FFPE) tissues and the fluorescent color products must be purchased separately from the kit. The v2 kit can also become 4-plex capable when used in combination with the RNAscope® 4-Plex Ancillary Kit which contains the block and development reagents for a fourth dye that are not contained in the standard v2 kit.

For those of you who are using automated in situ staining systems like the Leica Biosystems BOND RX or Ventana Discovery XT or ULTRA, there are specialized RNAscope kits designed to work with these systems. All of the kits I’ve described until now are meant for manual staining only. Note that there are currently no fluorescent kits available that are compatible with the Ventana systems.

Due to its simplicity and versatility, the RNAscope® 2.5 HD Brown kit is highly recommended for your first RNAscope assay if you are using human or rodent samples. Once you master the protocol using this assay, then you can switch fairly easily to the kit that best fits your needs. Just make sure you thoroughly read the protocol ahead of time for the small but important differences between kit procedures and required reagents.

Does Your Tissue Need a Spa Day? – Tissue Pretreatment Options

Just like being in a sauna is supposed to help open your pores, sometimes your tissue needs some spa treatment to be open enough for RNAscope® probes to find your target RNA. This permeability is achieved in two steps: 1) boiling your tissue with the RNAscope® Target Retrieval solution; and/or 2) treating your tissue with a protease digestion reagent.

The Target Retrieval step is necessary to undo the cross-linking that occurs from tissue fixation. If you are using fresh frozen tissue without fixation, then this step is unnecessary. The protease reagent used depends on the type of tissue and kit combination you are working with. Protease Plus is a mild protease treatment and is used with FFPE and fixed frozen tissue with the chromogenic kits. Protease III is a moderate protease treatment used with FFPE tissue, fixed frozen tissue, and cultured adherent and non-adherent cells with the fluorescent kits. Protease IV is the strongest protease treatment and is used with fresh frozen tissue with all kits.

Finally, if you want to use a chromogenic (brown or red) kit, then your tissue spa pretreatment will also need to include a hydrogen peroxide step to block endogenous peroxidase activity. Luckily, many of these reagents are included with the kits themselves, but be aware that certain combinations of kits and tissues will require additional reagents. For example, chromogenic kits come with RNAscope® Hydrogen Peroxide, RNAscope® Target Retrieval, and RNAscope® Protease Plus. This means if you are using anything other than FFPE and fixed frozen tissue with the chromogenic kit, you’ll need to buy the appropriate permeability reagent separately.

Don’t Give in to Analysis Paralysis

If all of this still feels pretty overwhelming, know that you aren’t alone. In fact, ACD Bio, the maker of RNAscope®, likely either experienced or anticipated a high level of confusion from users and has tried to mitigate this in several ways. Their website has several tables that aid in selecting the right assay kit and pretreatment reagents, as well as a way to generate a checklist for you based on your sample type and kit. In addition, I was assigned a technical specialist as a new user to help guide me through this entire planning and execution process. Reach out to them before purchasing supplies to make sure you get this personalized touch – I found it invaluable.

Once you know which kit and pretreatment reagents you need, the only things left to do are to select your RNA-specific hybridization probe and appropriate control probes. You’re almost there!

Additional Resources:

RNAscope® Pretreatment Reagents –

RNAscope® Manual Kits, Assays & Reagents (Chromogenic & Fluorescent) –

RNAscope® Multiplex Fluorescent Assay Options (More Detail On Fluorescent) –

RNAscope® Kits Specifically for Automated Systems – (Leica System), (Ventana System)

RNAscope® – Getting Started (includes checklist) –

Training Videos on RNAscope®  –

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Image Credit: Ryan Jeffs

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