RNAscope™ ISH - FAQs

• RNAscope is designed to detect any mRNA or ncRNA greater than 300 bases., although 1000 bases is optimal for a standard RNAscope probe design, which includes 20 ZZ pairs.
• BaseScope™ is designed to detect short target sequences that are 50 to 300 bases. The BaseScope probe is designed in 1-3 ZZ probe pairs
• miRNAscope™ targets highly abundant smRNAs that are at least 17 bases. 
• DNAscope™ targets chromosomal DNA with a minimum of 20kb and viral DNA integration with a minimum of 3kb. 

To run RNAscope assays in your lab, either manual or automated, 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 Bio-Techne's HybEZ™ II Hybridization System. This oven, unlike any other hybridization oven, provides humidity and temperature control that are necessary for proper RNAscope assay performance.

We recommend all first-time manual assay users to start with our RNAscope™ Introductory Pack. This provides required reagents you need to gain familiarity with the technique before performing your experimental studies. It also provides control slides to use as references to quality control RNA integrity and optimize sample pretreatment conditions for your studies. If you are interested in the automated assay on the Roche DISCOVERY ULTRA platform or the Leica Biosystems’ BOND RX, then Bio-Techne will provide you with an on-site training.

View our Getting Started page.

The main differences compared with an immunohistochemistry (IHC) workflow are the following:

• No cooling is required during epitope retrieval; users should directly put the slides in water at room temperature and proceed to protease step as per the manual Part 1.
• There is a protease digestion step for tissue permeabilization at the appropriate temperature. 
• The HybEZ oven is a must have instrument required for manual RNAscope hybridization as it maintains optimum humidity and temperature (40 °C) control.
• SuperFrost™ Plus slides are required for successful RNAscope assay. Other slide types may result in tissue detachment.
• ImmEdge Hydrophobic Barrier Pen (Catalog # 310018) is the recommended pen for maintaining the hydrophobic barrier throughout the RNAscope procedure.

Tissue fixation in accordance with standard clinical research guidelines is recommended. FFPE samples should be fixed in FRESH 10% NBF (neutral buffered formalin) for 16 – 32 h at RT. NOTE: Do not fix at 4 °C. Do not fix for less than 16 h or more than 32 h. Delayed fixation can degrade RNA and produce lower signal or no signal. Shorter time or lower temperature will result in under-fixation. For optimal results, we highly recommend using the 10% NBF tissue fixation methodology.

NOTE:  Recommended section thickness for FFPE samples is 5 +/- 1 μm, for fixed frozen tissue, it is 7 - 15 μm sections, and for fresh frozen tissue, it is 10 -  20 μm sections.

Running 3 slides minimum per sample is recommended: your target marker panel, a positive control, and a negative control probe.  The positive control will help determine whether the quality of RNA in the tissue specimen is sufficient for detecting your RNA target. The bacterial dapB negative control will help determine whether the tissue specimen is appropriately prepared for RNAscope and BaseScope assays.

For single-plex assays, PPIB is the suggested positive control for most tissues; for Duplex assay, two housekeeping genes, POLR2A and PPIB are provided as the positive controls. These probes are available in either RNAscope or BaseScope formats. For BaseScope, negative and positive control probes are available as either 1zz or 3zz’s; always use the appropriate zz control probes based on the number of zz’s of your target probe(s).

For the multiplex assays, the RNAscope™ 3-plex Positive Control Probe, targeting POLR2A, PPIB, and UBC, or the RNAscope™ 4-plex Positive Control Probe, targeting POLR2A, PPIB, UBC, and HPRT, are recommended. The RNAscope™ HiPlex12 Positive and Negative Control Probes are recommended for HiPlex assays.

For the miRNAscope assay, we recommend the use of miRNAscope™ Negative Control Probe - SR-Scramble-S1 and miRNAscope™ Positive Control Probe - SR-RNU6-S1 as appropriate controls.

Please note: our positive control probes are species specific. Only when the positive control has a score of 2+ for RNAscope or a 1+ for BaseScope, and the dapB negative control has a score of 0, can you confidently make a call on the expression of your target RNA in the tissue specimen. Similarly, for miRNAscope, a sample must have a positive control score of 3 and a negative control score of 0 to confidently interpret target RNA results within the sample.

• 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.
• Warm probes and wash buffer at 40 °C, precipitation occurs during storage and may affect the assay results.
• Retain adequate humidity in the Humidity Control Tray, to balance out temperature and humidity during hybridization.

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 RTU probe. If no specific C1 probe is used, then a Blank Probe Diluent (assay dependent) is used to dilute the probes.

To set up the assay, Bio-Techne recommends running 3 slides/sample, starting with one with your target probes using a C1 ready to use probe and mixing a channel C2, C3 and/or C4 target of your choice. The second slide should be the appropriate multiplex positive control probe and the third would be a dapB negative control probe. 

We recommend probe channels and fluorophores be selected with consideration of the expression levels of your targets. For example, a higher expression gene may be chosen for a wavelength with high autofluorescence in your sample (e.g. 488/green).

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 single plex Brown,  RNAscope 2.5 HD single plex Red or BaseScope v2 kits . These assays are designed to work only with C1 probes.

You have a few options:

• After sectioning of the tissue - FFPE slides can be stored with desiccant at room temp and used within 3 months. Frozen tissue slides can be stored at -80 °C in an airtight container for up to 3 months.
• After creating a hydrophobic barrier – FFPE section slides can be left to dry at room temperature overnight for use the following day.
• After probe hybridization – slides can be stored in 5X SSC buffer (not provided in the kit) overnight at RT. Before continuing with the assay, wash the slides twice with 1X Wash Buffer for 2 min at RT.

The RNAscope Multiplex Fluorescent v2 Assay, RNAscope Plus smRNA-RNA, and HiPlex v2 Assay can be imaged using either an epi-fluorescent or confocal microscope with the appropriate filters for assigned fluorophores. For specific excitation wavelength of each probe channel please refer to the appropriate user manual for your assay of interest.

RNAscope signal is shown as punctate dots. Each dot represents a single copy of an mRNA molecule.

There may be variation in dot intensity/size which is because of the differences in the number of ZZ probes bound to a target molecule. However, since each dot represents a single transcript, the number of dots is critical and not the intensity/size of the dot(s).

• 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.
• Warm probes and wash buffer at 40 °C, precipitation occurs during storage and may affect the assay results.
• Retain adequate humidity in the Humidity Control Tray, to balance out temperature and humidity during hybridization.

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 Bio-Techne's HybEZ™ II Hybridization System. This oven, unlike any other hybridization oven, provides humidity and temperature control that are necessary for proper RNAscope assay performance.

We recommend all first-time manual assay users to start with our RNAscope™ Introductory Pack. This provides required reagents you need to gain familiarity with the technique before performing your experimental studies. It also provides control slides to use as references to quality control RNA integrity and optimize sample pretreatment conditions for your studies. If you are interested in the automated assay on the Roche DISCOVERY ULTRA platform or the Leica Biosystems’ BOND RX, then Bio-Techne will provide you with on-site training.

The main differences compared with an immunohistochemistry (IHC) workflow are the following:

• No cooling is required during epitope retrieval; users should directly put the slides in water at room temperature and proceed to protease step as per the manual Part 1.
• There is a protease digestion step for tissue permeabilization at the appropriate temperature. 
• The HybEZ oven is a must have instrument required for manual RNAscope hybridization as it maintains optimum humidity and temperature (40 °C) control.
• SuperFrost™ Plus slides are required for successful RNAscope assay. Other slide types may result in tissue detachment.
• ImmEdge Hydrophobic Barrier Pen (Catalog # 310018) is the recommended pen for maintaining the hydrophobic barrier throughout the RNAscope procedure.

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 RTU Probe. If no specific C1 probe is used, then a Blank Probe Diluent (assay dependent) is used to dilute the probes.

To set up the assay, Bio-Techne recommends running 3 slides/sample, starting with one with your target probes using a C1 ready to use probe and mixing a channel C2, C3 and/or C4 target of your choice. The second slide should be the appropriate multiplex positive control probe and the third would be a dapB negative control probe. 

We recommend probe channels and fluorophores be selected with consideration of the expression levels of your targets. For example, a higher expression gene may be chosen for a wavelength with high autofluorescence in your sample (e.g. 488/green).

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 single plex Brown,  RNAscope 2.5 HD single plex Red or BaseScope v2 kits . These assays are designed to work only with C1 probes.

You have a few options:

• After sectioning of the tissue - FFPE slides can be stored with desiccant at room temp and used within 3 months. Frozen tissue slides can be stored at -80 °C in an airtight container for up to 3 months.
• After creating a hydrophobic barrier – slides can be left to dry at room temperature overnight for use the following day.
• After probe hybridization – slides can be stored in 5X SSC buffer (not provided in the kit) overnight at RT. Before continuing with the assay, wash the slides twice with 1X Wash Buffer for 2 min at RT.

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

Optimal sample pretreatment 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.

• RNAscope is designed to detect any mRNA or ncRNA greater than 300 bases., although 1000 bases is optimal for a standard RNAscope probe design, which includes 20 ZZ pairs.
• BaseScope is designed to detect short target sequences that are 50 to 300 bases. The BaseScope probe is designed in 1-3 ZZ probe pairs
• miRNAscope targets highly abundant smRNAs that are at least 17 bases. 
• DNAscope targets chromosomal DNA with a minimum of 20kb and viral DNA integration with a minimum of 3kb. 

Probes are designed with oligo pairs. Each oligo has two hybridizing regions, and ACD refers to these oligos as ZZ pairs. The “bottom” of each Z oligo comprises an 18 to 25-base region that is complementary to the target RNA. This sequence is selected for target-specific and uniform hybridization properties. Each ZZ oligo pair hybridizes to 36 to 50 bases of target RNA, and a typical RNAscope probe consists of 20 ZZ pairs spanning about 1000 bases of unique sequence. Redundancy and robustness are built into our design strategy resulting in high specificity.

The C1, T1 and S1 designation is the amplification channel the probe was designed to be detected in. The letter is used to designate which assay it can be used with. Any probe with a C listed is used with our RNAscope, BaseScope, and DNAscope assays; any probe with the T channel is used with our HiPlex assay. Any probe that has S1 in the probe names is designed for our miRNAscope assay. The RNAscope Plus Assay uses a single S1 probe with up to 3 C channels (C2, C3, and C4). 

Please note that when detecting multiple targets on the same sample, each target must have its own amplification channel assignment for compatibility and specificity of detection. 

We provide 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 our website. The exact probe pair location and sequences are considered Bio-Techne’s proprietary information.

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

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

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

However, note that manual and automated assays probes are formulated differently. Automated probes for use on Leica BOND RX and Roche DISCOVERY ULTRA systems are designated in our catalog with LS and VS, respectively. For example, LS-Hs-PPIB is for use on a Leica system instrument, and VS-Hs-PPIB is for use with the Roche system.

Our probe stability is tested for up to 2 years from the date of manufacturing when they are stored as recommended at 4 °C.

The RNAscope probe design algorithm examines each oligo probe in the probe set for potential cross-reactivity against the host transcriptome to ensure the probe specificity. There is a verification procedure following each major step during probe design to guarantee accuracy. We perform in-silico verifications and manufacture QC for RNAscope probes. The probe design algorithm selects oligos complementary to the input target sequence with compatible melting temperature for optimal hybridization at RNAscope assay condition and minimal cross-hybridization to off-target sequences. 

During the manufacture and formulation process, oligo synthesis QC is performed on each oligo probe. The oligo synthesis QC includes ESI mass spectrometry to ensure the probe sequences do not contain any deletions, additions or mutations, and OD reading to ensure correct concentration is provided upon delivery. 

 Finally, final inspection of finished good is conducted to ensure correct volume and label.

This depends on the sequence homology:  >95% homology is needed across species to create such probes. 

Please submit your request with your specifications on our webform. We will share feasibility results based on your specific requirements and target sequences.

RNAscope signal is shown as punctate dots. Each dot represents a single copy of an mRNA molecule.

There may be variation in dot intensity/size which is because of the differences in the number of ZZ probes bound to a target molecule. However, since each RNAscope dot represents a single transcript, the number of dots is critical and not the intensity/size of the dot(s).

RNAscope signal is detected as punctate dots. Clusters can result from overlapping signals from multiple mRNA molecules that are in close proximity to each other.

The RNAscope Multiplex Fluorescent v2 assay and HiPlex v2 assay can be imaged using either an epi-fluorescent or confocal microscope with the appropriate filters for assigned fluorophores. For specific excitation wavelength of each probe channel please refer to the appropriate user manual for your assay of interest.

Running 3 slides minimum per sample is recommended: your target marker panel, a positive control, and a negative control probe.  The positive control will help determine whether the quality of RNA in the tissue specimen is sufficient for detecting your RNA target. The bacterial dapB negative control will help determine whether the tissue specimen is appropriately prepared for RNAscope and BaseScope assays.

For single-plex assays, PPIB is the suggested positive control for most tissues; for duplex assay, two housekeeping genes, POLR2A and PPIB are provided as the positive controls. These probes are available in either RNAscope or BaseScope formats. For BaseScope, negative and positive control probes are available as either 1zz or 3zz’s; always use the appropriate zz control probes based on the number of zz’s of your target probe(s).

For the multiplex assays, the RNAscope™ 3-plex Positive Control Probe, targeting POLR2A, PPIB, and UBC, or the RNAscope™ 4-plex Positive Control Probe, targeting POLR2A, PPIB, UBC, and HPRT, are recommended. The RNAscope™ HiPlex12 Positive and Negative Control Probes are recommended for HiPlex assays.

For the miRNAscope assay, we recommend the use of miRNAscope™ Negative Control Probe - SR-Scramble-S1 and miRNAscope™ Positive Control Probe - SR-RNU6-S1 as appropriate controls.

Please note: our positive control probes are species specific. Only when the positive control has a score of 2+ for RNAscope or a 1+ for BaseScope, and the dapB negative control has a score of 0, can you confidently make a call on the expression of your target RNA in the tissue specimen. Similarly, for miRNAscope, a sample must have a positive control score of 3 and a negative control score of 0 to confidently interpret target RNA results within the sample.

You can analyze your image either semi-quantitatively or quantitatively. Please refer to the scoring guidelines for semi-quantitative analysis or use image analysis software to analyze quantitatively. Technical notes are available providing guidance on how you can use ImageJ/Cell Profiler/QuPath to quantify gene expression from your RNAscope images.  HALO software from Indica Labs is used in-house for quantitative analysis. 

Please contact Indica Labs directly at info@indicalab.com for HALO related questions.

RNA/protein co-detection is compatible with the RNAscope RED, RNAscope Multiplex Fluorescent, BaseScope v2, and miRNAscope (RED) assays.

Our sequential dual ISH/IHC workflow allows for IHC staining following RNAscope. Protease pretreatment is necessary for RNAscope but can impact your target protein of interest. Some epitopes may be better conserved than others after protease treatment and some optimization of protease may be necessary to find an optimal condition that allows for both RNAscope and IHC detection. The integrated co-detection workflow cross-links the primary antibody prior to the protease step, allowing conservation of the antigen/antibody for detection. 

The Co-detection Antibody Diluent (Catalog # 323160) has been formulated to ensure maximum retention of RNA sample quality.

Co-Detection Blocker prevents cross-detection of RNAscope signal by the IHC detection steps.

It is recommended that you use your preferred antibody clones for RNA-protein co-detection. For best results, titrate your preferred primary antibody concentrate in our Co-Detection Antibody Diluent (Catalog # 323160) in the context of the integrated co-detection workflow.

We always recommend starting by establishing a working IHC protocol and then using the same established secondary antibody for ICW. You may use any secondary that you have been using in the lab for your IHC workflow.  Please ensure that any secondary that you choose uses the appropriate detection chemistry (e.g. HRP-conjugated secondary for chromogenic RNAscope, and fluorescent-conjugated or HRP-conjugated secondary for fluorescent detection).

RNAscope, BaseScope and miRNAscope assays can be used with FFPE tissue, fresh-frozen tissue, fixed-frozen tissue, and cultured cells. DNAscope is only compatible with FFPE tissue samples. We have user manuals and protocols and recommendations for preparing each of these sample types. Note that cultured cell samples are not compatible with RNAscope HiPlex v2 assay.

Tissue fixation in accordance with standard clinical research guidelines is recommended. FFPE samples should be fixed in FRESH 10% NBF (neutral buffered formalin) for 16 – 32 h at room temperature.

NOTE: Do not fix at 4 °C. Do not fix for less than 16 h or more than 32 h. Delayed fixation can degrade RNA and produce lower signal or no signal. Shorter time or lower temperature will result in under-fixation. For optimal results, we highly recommend using the 10% NBF tissue fixation methodology.

NOTE: Recommended section thickness for FFPE samples is 5 +/- 1 μm, for fixed frozen tissue, it is 7-15 μm sections, and for fresh frozen tissue, it is 10-20 μm sections.

It is recommended that an RNAse-free environment is maintained while handling and sectioning tissues prior to fixation. Once the samples have been properly fixed, further RNA degradation is not expected to occur.

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.

Under-fixation of tissue specimens will result in protease over-digestion, which leads to loss of RNA and poor tissue morphology. Over-fixed tissue specimens will result in protease under-digestion, which leads to poor probe accessibility and low signal and signal/background ratio while maintaining excellent tissue morphology.

If you do not have information on how the tissue was prepared, or the tissues were prepared differently from our recommendations, you may need to vary one or both pretreatment steps (target retrieval and/or protease digestion. We recommend that tissues are tested along with our control slides (HeLa or 3T3) using positive (species-specific) and negative control probes. Observe the staining pattern and if the control slides are comparable to our positive and negative control image gallery, but your tissue samples show weak or low signal staining you will need to optimize the assay conditions. This entails identifying the optimum tissue pretreatment conditions by varying target retrieval, protease digestion, or both. For over-fixed tissues (tissue morphology looks good, but staining is weak), you will need to increase target retrieval and/or protease incubation times to increase probe accessibility. For under-fixed tissues (where tissue appears faded, with loss of cell borders) decrease the target retrieval and/or protease incubation times.