RNAscope Assay Workflow

Both temperature and humidity are critical to assay performance. The HybEZ™ oven is the only hybridization oven that ACD has extensively tested and validated. Other incubators/hybridization stations may not provide consistent results.

Protease digestion is also critical to assay performance. Under-digestion will result in lower signal and a ubiquitous background. Over-digestion will result in poor morphology and loss of RNA.

You have a few options, when you plan your assay-

C1 probe can be substituted with blank probe diluent and used with C2, C3 or C4 target probes for duplex/multiplex assay. Do not use channel C2 or channel C3 probes with RNAscope 2.5 HD singleplex Brown or 2.5 HD Red assays. These are designed to work only with C1 probes.

To independently detect target RNAs in a multiplex assay, each target probe must be in a different channel (C1, C2, C3, and/or C4) and one of the target probes must be in the C1 channel. Channel C1 target probes are Ready-To-Use (RTU), while channel C2, C3 and C4 probes are shipped as a 50X concentrated stock. The 50x probes for C2, C3, or C4 must be mixed with a C1 Ready to Use Probe. If no specific C1 probe is used, then a Blank Probe Diluent (assay dependent) is used to dilute the probes.

The main differences compared to IHC workflow are the following:

To run RNAscope assay in your lab for manual or automated assays you will need target probes, control probes, and a reagent kit. Both the manual and automated assays have control probes for common housekeeping genes which can be selected based on the expected expression level of your target (positive control probes are species specific). For the manual assay, a critical piece of benchtop equipment for routine success with RNAscope is ACD’s HybEZ Oven System (https://acdbio.com/hybez-system).

• Apply all amplifications steps in the right order; missing any step may result in no signal.
• For manual assay workflow, flick or tap the slides to remove residual reagent, however, do not let the slides dry at any time.
• Make sure the hydrophobic barrier remains intact so that the tissues do not dry at any time.
• Always use fresh reagents, this includes alcohol and xylene.
• Do not alter the protocol in any way, e.g. after boiling, do not cool down samples, they should go directly into dH20.