Please refer to the troubleshooting guide to assist you in optimizing your assay.
1. RNAscope™ ISH Overview
RNAscope™ Technology is a novel in situ hybridization (ISH) assay that detects target RNA within intact cells. The assay is based on a patented signal amplification and background suppression technology, representing a major advance over traditional RNA ISH. RNAscope™ Assays do not require an RNase-free environment.
The manual assay procedure can be completed in 7–8 hours or conveniently divided over two days. Most of the RNAscope™ Assay reagents are available in convenient Ready-To-Use (RTU) dropper bottles and provide a simple, nearly pipette-free workflow. The assay is also available for automation on the Ventana DISCOVERY™ XT or ULTRA, or the Leica Biosystems’ BOND RX system.
The RNAscope™ protocol is similar to immunohistochemistry (IHC) with a few key differences as described in the following section
2. Key Differences between RNAscope™ ISH and IHC
Antigen retrieval conditions often require optimization in both RNAscope™ and IHC, depending on the tissue type and how the sample was fixed and processed. We recommend optimizing conditions for the RNAscope™ Assay if tissue samples are not prepared according to the ACD recommended guideline of fixing samples in fresh 10% NBF for 16–32 hours.
The main differences compared to an IHC workflow are the following:
- No cooling is required during antigen retrieval. Directly place the slides in water at room temperature to immediately stop the reaction and proceed to the next step as described in the user manual.
- A protease digestion step is included to permeabilize tissue. Ensure that the temperature is maintained at 40°C during this step.
- The HybEZ™ Hybridization System maintains optimum humidity and temperature during the assay workflow and is required for RNAscope™ hybridization steps.
- Superfrost® Plus slides are required. Other slide types may result in tissue detachment.
- Xylene based mounting media (such as CytoSeal XYL) is required for the RNAscope™ 2.5 HD Brown assay.
- EcoMount (Biocare Medical) or PERTEX® (HistoLab®) media is required for the RNAscope™ 2.5 HD Red and 2-plex assays. No other mounting media should be used.
- Diluting Gill’s Hematoxylin I 1:2 is suggested for counter staining
- ImmEdge® Hydrophobic Barrier Pen (Vector Laboratories Cat. No. 310018) is the only pen that will maintain a hydrophobic barrier throughout the RNAscope™ procedure. No other barrier pen should be used.
3. Key RNAscope™ Assay Guidelines
Before you get started refer to our Getting Started Page.
- 01 Pointers for the RNAscope™ manual assays
- Follow the protocol exactly as described in the user manual. For manual assays, user manuals are divided into two parts: sample preparation (Part 1), and detection (Part 2). Refer to the User Manual Selection Guide.
- Before beginning the assay, review sample pretreatment recommendations. These are critical steps for accessing the RNA in your tissue sample.
- Always run positive and negative reference probes (e.g. PPIB and dapB respectively) on your sample to assess sample RNA quality and optimal permeabilization.
- Please make sure you have all the required materials, especially each of the following:
- Immedge pen (Vector Laboratories)
- Superfrost Plus slides (Fisher Scientific)
- Fresh reagents (ethanol and xylene)
- Fresh 10% NBF (neutral-buffered formalin)
- Tissue-Tek® slide rack and staining dishes or the ACD EZ Batch™ Slide System
- Hotplate, drying oven, water bath, thermometer, microscope, etc.
- HybEZ™ Hybridization System, required for RNAscope™ hybridization to maintain optimum humidity and temperature
Workflow guidelines:
- Apply all amplifications steps in the right order; missing any step will result in no signal.
- Flick or tap the slides to remove residual reagent, but do not let the slides dry out at any time.
- Make sure the hydrophobic barrier remains intact so that the tissues do not dry out.
- Always use fresh reagents, including ethanol and xylene.
- Do not alter the protocol in any way.
- Warm probes and wash buffer at 40°C. Precipitation occurs during storage and may affect the assay results.
- Maintain adequate humidity by keeping the humidifying paper wet in the Humidity Control Tray
For the RNAscope® 2.5 HD - RED Assay
- Only use EcoMount or PERTEX as the mounting media for the RNAscope™ 2.0 HD Red detection assay.
For the RNAscope® 2-plex Chromogenic Assay
- Channel C1 target probes are Ready-To-Use (RTU), while channel C2 probes are shipped as a 50X concentrated stock. To independently detect target RNAs in a 2-plex assay, the target probes must be in different channels and there must be a C1 probe in the mixture. A “Blank Probe – C1” (Cat. No. 300041) can be used if no C1 probe is being included in the assay
Components Mixing Ratio Probes C2:C1 1:50 Probes C2:C1 1:50 Probes C2:C1 1:50 Probes C2:C1 1:50 - 02 Pointers for the RNAscope™ automated assays
For the Ventana DISCOVERY XT or ULTRA Systems
Troubleshoot the instrument
Check instrument maintenance:
- Refer to the user manual for instrument maintenance.
- Call your Ventana/ Roche Diagnostics representative to perform the decontamination protocol every three months to prevent microbial growth in the lines.
- Call your Ventana/ Roche Diagnostics representative to replace all bulk solutions with the recommended buffers before running the RNAscope™ assay. Rinse the containers thoroughly and make sure the internal reservoir has been purged several times with the appropriate buffer.
- If you use water to clean the instrument, make sure residual water is replaced with the appropriate buffers by purging several times.
Optimize software settings:
- Uncheck the Slide Cleaning option.
- If you are working with software version 2.0, the fully automated setting is applicable only when working with brain and spinal cord samples.
- Refer to the user manual for hybridization temperatures. Do not make any adjustments to the recommended temperatures unless otherwise instructed by ACD’s technical support.
Troubleshoot reagents
- Before beginning the assay, refer to the user manual for tissue pretreatment times and recommended guidelines.
- For over- or under-fixed tissues, adjust the Pretreat 2 (boiling) and/or protease treatment times (Pretreatment A and B). Refer to the user manual.
- Always run positive and negative controls (PPIB and dapB respectively), to qualify your sample and check assay performance.
- Use the DISCOVERY 1X SSC Buffer only and dilute 1:10 prior to adding the buffer to the optional bulk buffer container. Do not use the Benchmark 10X SSC Buffer.
- Use the RiboWash Buffer diluted 1:10 in the RiboWash bulk container only.
For Leica Biosystems’ BOND RX System
- The recommended standard tissue pretreatment is 15 minutes Epitope Retrieval 2 (ER2) at 95°C and 15 minutes Enzyme (Protease) at 40°C.
- For a milder pretreatment, the recommended conditions are 15 min ER2 at 88°C and 15 min Protease at 40°C.
- For extended pretreatment times, increase the ER2 time in increments of 5 minutes and increase the Protease time in increments of 10 minutes, while keeping the temperatures constant (e.g. 20 min ER2 at 95°C and 25 min Protease at 40°C; 25 min ER2 at 95°C and 35 min Protease at 40°C). You may also apply this process to over-fixed tissues.
- The “Mock probe” and “Bond wash” Open containers are user-filled with 1x Bond Wash Solution.
- The RNAscope™ 2.5 LS Brown and LS Red assays utilize Leica Biosystems’ Bond Polymer Refine Detection and Bond Polymer Refine Red Detection kits, respectively. Do not use any other chromogen kits.
- Do not alter the staining protocol in any way. The parameters in the staining protocol have been optimized to run RNAscope on the instrument. You may change the hematoxylin incubation time according to your needs.
4. RNAscope™ Recommended Workflow
If sample preparation conditions do not match recommended guidelines or are unknown, we strongly recommend qualifying your samples prior to performing any experiments. See Figure 1 for the recommended workflow
1. Run samples along with the control slides provided by ACD (Cat. No. 310045 for Human Hela Cell Pellet, and Cat. No. 310023 for Mouse 3T3 Cell Pellet) using ACD positive and negative control probes.
- The ACD Positive Control Probes include three different housekeeping genes to test tissue RNA integrity. You may use the low-copy (10–30 copies per cell) housekeeping genes Cyclophilin B (PPIB) or the low copy (5–15 copies) Polymerase (RNA) II (DNA directed) polypeptide A (POLR2A), or high copy Ubiquitin C (UBC).
- The ACD Negative Control Probe uses bacterial dapB and should not generate signal in properly fixed tissue.
2. Use the RNAscope™ scoring guidelines to evaluate staining results.
- Successful PPIB staining should generate a score ≥2 and UBC score ≥3 with relatively uniform PPIB/POLR2A/UBC signal throughout the sample.
- Samples should display a dapB score of <1 indicating low to no background.
3.Use the control slides as a reference to determine if the RNAscope assay was performed correctly. See Figure 2 for RNAscope scoring guidelines.
4. Depending on staining results, you may need to optimize pretreatment conditions for your samples.
Figure 1. Recommended RNAscope® workflow to test samples prior to evaluating target gene expression.
5. Scoring Guidelines
The RNAscope Assay uses a semi-quantitative scoring guideline to evaluate the staining results. When interpreting RNAscope staining we recommend scoring the number of dots per cell rather than the signal intensity. The number of dots correlates to the number of RNA copy numbers, whereas dot intensity reflects the number of probe pairs bound to each molecule.
An example of how to develop such a guideline for semi-quantitative assessment of RNAscope staining intensity is presented below for a gene with expression level varying between 1 to > 15 copies per cell (PPIB). If your gene expression level is higher or lower than this range, you may need to scale the criteria accordingly
Figure 2. RNAscope Scoring Guidelines on HeLa control slides at 20X magnification.
| Score | Criteria |
|---|---|
| 0 | No staining or <1 dot/ 10 cells |
| 1 | 1-3 dots/cell |
| 2 | 4-9 dots/cell. None or very few dot clusters |
| 3 | 10-15 dots/cell and <10% dots are in clusters |
| 4 | >15 dots/cell and >10% dots are in clusters |
*If <5% of cells score 1 and >95% of cells score 0, a score of 0 will be given. If 5-30% of cells score 1 and >70% of cells score 0, a score of 0.5 will be given. Scoring is performed at 20X magnification.
- 01Interpreting RNAscope results
Compare the expression of your target gene with both negative (dapB) and positive controls (PPIB, UBC, or POLR2A). Successful staining should have a PPIB/POLR2A score ≥2 or UBC score ≥3 and a dapB score <1. See Figure 3 for an example.
Figure 3. RNAscope assay results comparing a high expressing HR-HPV case study with a positive and a negative control.
A) HPV-HR18 B) UBC Positive Control C) DapB Negative Control
6. Sample Preparation
Sample preparation is critical for successful staining of tissue with RNAscope ISH methodology. The RNAscope manual assays can be used with FFPE (formalin-fixed, paraffin-embedded), cultured cells, fresh-frozen, fixed frozen tissues, or PBMC (Peripheral Blood Mononuclear cells). Please refer to the appropriate sample preparation guides as shown in the User Manual Selection Guide to ensure your samples are prepared correctly for the detection assay.
Note: Tissue thickness for fixed frozen tissue should be between 7–15 µm and 10–20 µm for fresh frozen tissue. Use Fisher Scientific SuperFrost Plus Slides for all tissue types to avoid tissue loss.
- 01Pointers for preparing FFPE tissue
Prepare tissues according to standard methods:
- Tissue specimens should be fixed in fresh 10% NBF for 16–32 hours at room temperature and blocked into a thickness of 3–4 mm.
Note: Under-fixation will result in significant RNA loss during storage and may result in low signal when performing the RNAscope Assay.
- Dehydrate in a graded series of ethanol and xylene, followed by infiltration by melted paraffin held at no more than 60°C
- Trim paraffin blocks as needed and cut embedded tissue into 5 +/- 1 µm sections using a microtome.
- Place paraffin ribbon in water bath, and mount sections on SuperFrost Plus Slides.
- Air dry slides overnight at room temperature. Do not bake slides unless they will be used within one week.
When tissue preparation method is unknown:
In many situations information on tissue preparation procedures may be unavailable. Tissue optimization steps depend not only on the type of tissue, but also the age of the animal. In particular if you were about to perform in situs on embryonic tissue, each developmental stage may require differential treatment. Simple optimization steps can help obtain quality data. Optimal conditions are dependent on tissue type, age, and fixation. - Tissue specimens should be fixed in fresh 10% NBF for 16–32 hours at room temperature and blocked into a thickness of 3–4 mm.
7. Optimize the Assay
In IHC, antigen retrieval conditions often require optimization and depend on the tissue type and how the sample was fixed and processed. RNAscope target retrieval conditions may also need optimization, particularly if tissue samples were not fixed for 16–32 hours in fresh 10% neutral-buffered formalin (NBF) at room temperature.
Guidelines to follow:
- Always test representative samples with positive and negative control probes. Positive controls should give a uniform signal, while negative controls should display little to no signal.
Note: A list of control probes for both manual and automated assays are available from ACD.
- Evaluate the positive and negative staining results of your samples following our evaluation guidelines (see V. Scoring Guidelines). If results are acceptable, no further optimization is needed. Otherwise, adjustment of pretreatment conditions may be required to obtain optimal results.
8. Troubleshooting Staining Patterns
- 01Staining examples for various pretreatment digestion conditions
Note: When working with RNAscope 2.5 HD assays or using the new pretreatments for optimization purpose, please use combination of Target Retrieval and Protease Plus to replace Pretreatment 2 and Pretreatment 3.
Possible Morphology
PatternExample Morphology Description Possible Effects Under-digested 
Xenograft tissue, dapB (8 min P2 + 15 min P3)
Xenograft tissue, Hs-PPIB (8 min P2 + 15 min P3)
Excellent morphology, strong hematoxylin staining Weak/no signal due to poor probe accessibility; nuclear background in liver/kidney tissue Over-digested 
Xenograft tissue, dapB (15 min P2 + 30 min P3)
Xenograft tissue, Hs-PPIB (15 min P2 + 30 min P3)
Destroyed tissue morphology, Doughnut/ghost nuclei, weak hematoxylin staining High background, nonuniform strong/weak signal Optimal Digestion 
Xenograft tissue, dapB (8 min P2-weak boil + 30 min P3)
Xenograft tissue, Hs-PPIB (8 min P2-weak boil + 30 min P3)
Intact Tissue morphology and nuclei. Homogenous hematoxylin staining High signal to noise ratio. Strong staining for positive controls with no/negligible background - 02Staining Patterns
Issue Probable Cause Suggested Action No
stainingSuboptimal tissue preparation (overfixation**/under-fixation*) Prepare tissue samples according to ACD recommended procedures. The best pretreatment conditions are listed in user manuals and tech notes for
specific sample types and are available at: User Manual Selection Guide To optimize pretreatment conditions (boiling and protease digestion times):- Gradually increase boiling/protease time for over-fixed tissues (Figures 4 and 5). For example, vary the boiling time from 15 minutes to 20, 25, and 30 minutes.
- Decrease boiling/protease time for under-fixed tissues (Figures 4 and 5). For example, vary incubation time from 30 minutes to 25, 20, and 15 minutes.
Hybridization temperature not optimal - Use HybEZ oven when performing the RNAscope assay for optimal results
- Ensure HybEZ Oven is at 40oC for the length of the assay unless otherwise noted in the user manual
Reagents are used in the wrong sequence Apply reagents in the correct order Gene of interest not expressed Check positive control to confirm the technical accuracy of the assay
**Over-fixed/under-digested tissue: Tissue morphology looks excellent with weak/no signal and low signal/background ratio due to poor probe accessibility.
*Under-fixed/over-digested tissue: Poor tissue morphology (tissue appear faded with loss of cell borders), loss of RNA due to protease over-digestion.
9. Troubleshooting High Background
| Issue | Image of Issue | Probable Cause | Suggested Action | Corrected Staining | |
|---|---|---|---|---|---|
| Nuclear background |  Standard pretreatment on Mm kidney –dapB, nuclear background observed | Pretreatment conditions not optimal
| Optimize pretreatment conditions
|  7 min P2 on Mm kidney - dapB, clean background | |
| Nuclear hazy background (Leica BOND RX) |  Human Tonsil: 15 min P2 + 15 min P3 (image represents RNAscope LS Bond RX staining) | Under-pretreatment | The presence of nuclear background staining (entire nuclei stained brown) can sometimes be removed by increasing pretreatment times. Increase the ER2 time in increments of 5 min and increase the protease time in increments of 10 min while keeping the temperature constant. You may also apply this process to over-fixed tissues. |  Human Tonsil: 20 min P2, 20 min P3 (image represents RNAscope LS Bond RX staining), clean background
| |
| Extracellular background | Incomplete paraffin removal | Use fresh/unused EtOH and Xylene and agitate slides during incubation steps | |||
| Suboptimal tissue preparation | Prepare tissue samples according to ACD recommended procedures | ||||
| Cytoplasmic background |  HeLa cell pellet, dapB; Dried out tissue, cytoplasmic background | Tissue dries up during assay |
|  HeLa cell pellet, dapB; No drying between Amp steps, clean background | |
**Over-fixed/under-digested tissue: Tissue morphology looks excellent with weak/no signal and low signal/background ratio due to poor probe accessibility.
*Under-fixed/over-digested tissue: Poor tissue morphology (tissue appear faded with loss of cell borders), loss of RNA due to protease over-digestion.
10. Troubleshooting Other Issues
| Issue | Image of Issue | Probable Cause | Suggested Action | Corrected Staining |
|---|---|---|---|---|
| Tissue detaches from slides | Wrong slides used | Use only SuperFrost® Plus slides avoid tissue sliding off, Fischer Scientific, Cat. No. 12-550-15 | ||
 | Suboptimal tissue preparation |
| ||
| Unknown tissue preparation method | Sample provider/clinical site/vendor did not provide detailed instructions | Follow the appropriate Tissue Specimen Preparation and Assay Optimization Guideline to determine whether your tissue specimen is appropriately fixed and processed
Refer to VII. Optimize the Assay on page 9.
Please select appropriate user manuals for the tissue and assay type you are working with. The optimal pretreatment conditions for each assay and tissue type are located in user manuals and tech notes listed in the following document. User Manual Selection Guide |
11. Additional References
- Training videos are available at Training videos.
- For User Manuals, use the manual selection guide available at User Manual Selection Guide.
- Image Gallery is available at Image Gallery.
- Technology Whitepaper is available at Technology Whitepaper.
- For publications, go to Publications to find an RNAscope publication relevant to your research or to view images from publications.
Top Frequently Asked Questions
- What is the minimum sequence length of the target region required for RNAscope probe pool design?
RNAscope is designed to detect any mRNA or ncRNA greater than 300 bases. A standard RNAscope probe design 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 detects RNAs that are between 17 to 50 bases.
- What do I need to get started?
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). This oven, unlike any other hybridization oven, provides humidity and temperature control that are necessary for proper RNAscope assay performance.
ACD recommends 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 Ventana Discovery ULTRA platform or the Leica Biosystems BOND RX, then ACD will provide you with an on-site training.
- What are the key differences between RNAscope ISH versus an IHC workflow?
The main differences compared to 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.
- HybEZ 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.
- Freshly prepared Xylene based (such as CytoSeal XYL) mounting media is required for the RNAscope 2.5 HD Brown assay.
- Ecomount Mounting (recommended) or Vectamount media are required for the RNAscope 2.5 HD Red assay. No other mounting media should be used.
- Counter staining: diluting 1:2 Gill’s Hematoxylin I is required for counter staining
- ImmEdge Hydrophobic Barrier Pen (P/N 310018) is the recommended pen for maintaining the hydrophobic barrier throughout the RNAscope procedure.
- How should my tissue be fixed? Can I use 4% (paraformaldehyde) PFA instead of 10% NBF?
ACD recommends tissue fixation in accordance with standard clinical research guidelines. FFPE samples should be fixed in FRESH 10% NBF (neutral buffered formalin) for 16 – 32 hrs at RT. NOTE: Do not fix at 4°C. Do not fix for < 16 hrs or > 32 hrs. 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, ACD highly recommends 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.
- What controls are recommended to interpret results?
ACD always recommends running 3 slides minimum per sample: 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 singleplex 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; please use the appropriate zz control probes based on the number of zz’s of your target probe(s).
For 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 Control Probe is recommended for HiPlex assays.
For the miRNAscope assay, ACD recommends the use of miRNAscope™ Negative Control Probe - SR-Scramble-S1 and miRNAscope™ Positive Control Probe - SR-RNU6-S1 as appropriate controls.
Please note that 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, you can 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.
- ACD recommended tips and tricks when performing the RNAscope assay workflow?
- 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.
- How do I set up my probes for RNAscope multiplexing?
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.
To set up the assay, ACD recommends to run 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.
ACD recommends 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).
- Can I omit my C1 probe from the multiplex/duplex assay? How do I use the blank probe?
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.
- Are there stopping points if I can’t complete the manual assay in one day?
You have a few options, when you plan your assay-
- 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 over night 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.
- Can my fluorescent microscope work with RNAscope assay?
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.
- What is a typical RNAscope signal and what does a dot mean?
RNAscope signal is shown as punctate dots. Each dot represents a single copy of an mRNA molecule.
- What is the significance of dot size?
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).
RNAscope Assay Workflow
- ACD recommended tips and tricks when performing the RNAscope assay workflow?
- 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.
- What do I need to get started?
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). This oven, unlike any other hybridization oven, provides humidity and temperature control that are necessary for proper RNAscope assay performance.
ACD recommends 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 Ventana Discovery ULTRA platform or the Leica Biosystems BOND RX, then ACD will provide you with an on-site training.
- What are the key differences between RNAscope ISH versus an IHC workflow?
The main differences compared to 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.
- HybEZ 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.
- Freshly prepared Xylene based (such as CytoSeal XYL) mounting media is required for the RNAscope 2.5 HD Brown assay.
- Ecomount Mounting (recommended) or Vectamount media are required for the RNAscope 2.5 HD Red assay. No other mounting media should be used.
- Counter staining: diluting 1:2 Gill’s Hematoxylin I is required for counter staining
- ImmEdge Hydrophobic Barrier Pen (P/N 310018) is the recommended pen for maintaining the hydrophobic barrier throughout the RNAscope procedure.
- How do I set up my probes for RNAscope multiplexing?
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.
To set up the assay, ACD recommends to run 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.
ACD recommends 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).
- Can I omit my C1 probe from the multiplex/duplex assay? How do I use the blank probe?
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.
- Are there stopping points if I can’t complete the manual assay in one day?
You have a few options, when you plan your assay-
- 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 over night 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.
- What are the most critical factors affecting assay performance?
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.
Probe Design
- What is the minimum sequence length of the target region required for RNAscope probe pool design?
RNAscope is designed to detect any mRNA or ncRNA greater than 300 bases. A standard RNAscope probe design 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 detects RNAs that are between 17 to 50 bases.
- What is a Z?
Probes are designed with oligo pairs. Each oligo has two hybridizing regions, and ACD refers to these oligos as ZZ pairs. The “bottom” of the Z oligo has 18 to 25-base region that is complementary to the target RNA. This sequence is selected for target specific hybridization and uniform hybridization properties. So, 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.
- What is the difference between a C1, T1 or S1 probe?
The C1, T1 and S1 designation is the amplification channel the probe was designed to be detected in. The letter is used to designate the assay it can be used with. Any probe with a C listed is used with our RNAscope and Basescope assays; any probes with the T designation is used with our HiPlex assay. Any probe that has the S1 in the probe names is designed for our miRNAscope assay.
In the fluorescent assay, the amplification channel number (for example: C2, C3, C4 or T1, T2, T3, etc.) allows you to multiplex your targets to be detected using various fluorophores.
- Can ACD provide probe sequence information or oligo pair locations?
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.
- Can I use probes across different RNAscope manual detection assays?
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.
Note, however, that manual and automated assays probes are formulated differently. Automated probes for use on Leica and Ventana are designated in our catalog with LS and VS, respectively. For example, LS-Hs-PPIB = Leica System automated assay probe, and VS-Hs-PPIB = Ventana System automated assay probe.
- How stable are RNAscope probes?
Our probe stability is tested for up to 2 years from the date of manufacturing when they are stored as recommended at 4°C.
- What is ACD’s process for probe quality control, is each probe tested?
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. ACD does not validate probes, primarily because all probes are nucleic acids, and all probes adhere to strict rules of hybridization. Therefore, the precise melting temperature of each oligo comprising a probe is known and each oligo is tested in silico and no other transcribed sequence.
- Can ACD design a probe that cross detects across 2 or more species?
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
RNAscope Assay Visualization and Interpretation
- What is a typical RNAscope signal and what does a dot mean?
RNAscope signal is shown as punctate dots. Each dot represents a single copy of an mRNA molecule.
- What is the significance of dot size?
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).
- What is the difference between a dot and a cluster?
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.
- Can my fluorescent microscope work with RNAscope assay?
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.
- What controls are recommended to interpret results?
ACD always recommends running 3 slides minimum per sample: 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 singleplex 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; please use the appropriate zz control probes based on the number of zz’s of your target probe(s).
For 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 Control Probe is recommended for HiPlex assays.
For the miRNAscope assay, ACD recommends the use of miRNAscope™ Negative Control Probe - SR-Scramble-S1 and miRNAscope™ Positive Control Probe - SR-RNU6-S1 as appropriate controls.
Please note that 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, you can 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.
- How do I analyze my images to quantify gene expression?
You can analyze your image either semi-quantitatively or quantitatively. Please refer to the scoring guideline for semi-quantitative analysis or use image analysis software to analyze quantitatively. ACD has technical notes providing guidance on how you can use ImageJ/Cell Profiler/QuPath to quantify gene expression from your RNAscope images. In-house ACD uses HALO software from Indica Lab for quantitative analysis.
- How can I get more information about HALO software?
Please contact Indica Labs directly at info@indicalab.com for HALO related questions.
RNA/Protein Co-Detection
- Is my ISH assay compatible with the ACD Integrated Co-Detection Workflow using the RNA-Protein Co-Detection Ancillary kit?
RNA/protein co-detection is compatible with the RNAscope 2.5 HD RED, RNAscope Multiplex Fluorescent V2, BaseScope v2, and miRNAscope HD (RED) assays.
- How does the RNA/protein integrated co-detection workflow (ICW) differ from the dual ISH/IHC sequential workflow?
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 new integrated co-detection workflow cross-links the primary antibody prior to the protease step, allowing conservation of the antigen/antibody for detection. This workflow increases the compatibility of RNA-protein co-detection across a wide variety of antibodies.
- Why do I need to use a special Antibody Diluent from ACD?
The ACD Antibody diluent has been formulated to ensure maximal retention of RNA sample quality.
- What is the purpose of Co-Detection Blocker?
Co-Detection Blocker prevents cross-detection of RNAscope signal by the IHC detection steps.
- Does ACD have specific antibody clones or part numbers to recommend for use with the RNA/protein integrated co-detection workflow?
With the increased compatibility of the new integrated co-detection workflow across a wide variety of antibodies, there are no specific antibody clone recommendations. ACD encourages you to use your preferred clones for RNA-protein co-detection. For best results, titrate your preferred primary antibody concentrate in ACD’s Co-Detection Antibody Diluent in the context of the integrated co-detection workflow.
- What secondary is recommended for IHC detection?
ACD always recommends starting by establishing a working IHC protocol and then using the same established secondary for ICW. You may use any secondary 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 ICW, fluorescent-conjugated or HRP-conjugated secondary for fluorescent detection).
Sample Preparation
- Which sample types are compatible with RNAscope?
RNAscope, BaseScope and miRNAscope assays can be used with FFPE tissue, fresh-frozen tissue, fixed-frozen tissue, and cultured cells. ACD has user manuals and technical notes and recommendations for preparing each of these sample types. Note that cultured cell samples are not compatible with RNAscope HiPlex v2 assay.
- How should my tissue be fixed? Can I use 4% (paraformaldehyde) PFA instead of 10% NBF?
ACD recommends tissue fixation in accordance with standard clinical research guidelines. FFPE samples should be fixed in FRESH 10% NBF (neutral buffered formalin) for 16 – 32 hrs at RT. NOTE: Do not fix at 4°C. Do not fix for < 16 hrs or > 32 hrs. 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, ACD highly recommends 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.
- Should I use DEPC-treated and specified RNAse-free reagents (e.g., RNAse-free PBS, and 4% PFA made with RNAse-free PBS) when preparing my samples?
ACD recommends maintaining RNAse-free environment while handling and sectioning tissues prior to fixation. Once the samples have been properly fixed, further RNA degradation is not expected to occur.
- Is there a specific protocol for TMAs (tissue microarrays)?
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.
- What is the impact of under-fixation or over-fixation of FFPE tissue specimens on RNAscope assay?
Under-fixation of tissue specimens will result in protease over-digestion, which leads to loss of RNA and poor tissue morphology. Over-fixed tissue specimen will result in protease under-digestion, which leads to poor probe accessibility and low signal and signal/background ratio while maintaining excellent tissue morphology.
- I am not sure how the tissues were prepared? What pretreatment conditions should I use?
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. Observe the staining pattern and if the control slides are comparable to ACD’s positive and negative image gallery, but your tissues 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.
