Faqs

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.

Yes, RNAscope works just as well on properly fixed and prepared TMAs as it does on individual tissue sections. Because there may be variability from core to core in the TMA, optimization of the pretreatment conditions may be required.

ACD understands that in some situations, you may not have information on how the tissue was prepared or the tissues were prepared differently from our recommendations. In that case, you may need to vary one or both pretreatment steps (target retrieval and/or protease digestion). ACD recommends that tissues are tested along with ACD control slides (Hs or Mm) using positive and negative control probes.

This depends on the sequence homology:  >95% homology is needed across species to create such probes. Please contact your local ACD account executive for information specific to your gene. If you are a new customer and would like to get started please use contact us link on our website (http://www.acdbio.com/about/contact). ACD will get back to you with the requested information

ACD probe design algorithm is validated in silico to select oligos with compatible melting temperatures for optimal hybridization at RNAscope assay conditions and minimal cross-hybridization to off-target sequences. There is a verification procedure following each major step during probe design to guarantee the accuracy. Please refer to the RNAscope technique paper for details: http://www.ncbi.nlm.nih.gov/pubmed/22166544.

Yes. The same channel 1 (C1) Ready-to-Use probe can be used in each manual assay format – single-plex for chromogenic, 2-plex chromogenic and fluorescence multiplex. 

With the duplex assay, you will need to use channel 1 (C1) and channel 2 (C2). 

With the multiplex assay (for example: 4-plex), you will need to use channel 1 (C1), channel 2 (C2), channel 3 (C3) and channel 4 (C4) probes. 

ACD provides the 5’ and 3’ nucleotide positions of the target probe region and the number of probe pairs generated to that region. This information is available as you review your probe information on the ACD website. The exact probe pair location and sequences are considered ACD’s proprietary information.