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:

    1. 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.
       

    2. Dehydrate in a graded series of ethanol and xylene, followed by infiltration by melted paraffin held at no more than 60°C
    3. Trim paraffin blocks as needed and cut embedded tissue into 5 +/- 1 µm sections using a microtome.
    4. Place paraffin ribbon in water bath, and mount sections on SuperFrost Plus Slides.
    5. 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.

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
    Pattern
    Example 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
    staining
    Suboptimal 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

  • Tissue is overdigested*
  • Tissue is underdigested**

Optimize pretreatment conditions

  • Decrease boiling and/or protease if tissue is over-digested*
  • Increase boiling and/or protease if tissue is under-digested**

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
  • Completely cover tissue when applying reagents
  • Keep humidifying paper moist inside the HybEZ Slide Tray
  • Process slides one at a time to prevent drying
  • Make sure slides are kept level during incubations
  • Ensure HybEZ Oven is at the appropriate temperature

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
  • Prepare tissue samples according to recommended procedures
  • Bake slides for a longer time (up to overnight)
  • Reduce boiling time


Baked for 1 hour at 60°C

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

  • Run a preliminary assay with positive and negative reference genes-> look at the tissue morphology-> if results optimal (high signal/noise ratio and no background) proceed to marker staining OR

Refer to VII. Optimize the Assay on page 9.

  • Adjust the incubation times accordingly based on how the tissue morphology looks from preliminary experiment
  • Adjust protease dilution etc.

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

Top Frequently Asked Questions

  • Can my fluorescent microscope work with RNAscope assay? For fluorescent multiplex assays, what are the probe channels and what are the excitation emission wavelengths of the fluorophores?

    The RNAscope® Fluorescent kits are primarily targeted for fresh frozen and cultured cells. This is mainly due to imaging and analysis challenges with interference from tissue autofluorescence. We recommend the Leica DM series or equivalent or Zeiss Axio Imager. You can run the RNAscope® Fluorescent kit on solid tumor FFPE tissues if you have access to a multi-spectral imaging system, such as Nuance FX (Nuance) and Vectra from PerkinElmer. For specific excitation wavelength of each probe channel please refer to page 7 of our RNAscope® Fluorescent Multiplex Kit User Manual PART 2 which can be found here http://www.acdbio.com/technical-support/downloads/technical-doc/rnascope-mui-fluorescent-user-manual/

  • How do I quantify RNAscope data/staining?

    RNAscope detects individual RNA molecules, which appear as distinct dots, you can use semi-quantitative RNAscope scoring e.g., 1+ to 4+ based on number of dots per cell to interpret staining patterns. We also provide for chromogenic single-plex staining RNAscope® SpotStudio™ v1.0 Software, advanced image analysis solution designed to quantify RNA molecules (“dots”) on a cell-by-cell basis in stained fixed tissue.
    Please visit our website for more information: http://www.acdbio.com/products/spotstudio-software

  • How do I interpret RNAscope data/staining?

    We always recommend running 3 slides per sample, your target marker a positive & a negative control probe. Retrospectively collected tissue specimens may not be prepared according to the Specimen Preparation Guideline. Their RNA quality is also unknown. The UBC (POLR2A for RNAscope® 2.0) positive control will help determine whether the RNA in the tissue specimen is of sufficient quality for detecting your RNA biomarker. The bacterial dapB negative control will help determine whether the tissue specimen is appropriately prepared. Only when the UBC positive control has a score of 3+/4+ (POLR2A 1+) and the DapB negative control has a score of 0/1+, you can confidently make a call on the expression of your target RNA in the tissue specimen

  • 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, 2x.
    • 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. For the manual assay, a critical piece of benchtop equipment for routine success with RNAscope is ACD’s HybEZ System (http://www.acdbio.com/products/hybez). 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 XT or ULTRA platform or the Leica Biosystems BOND RX, then we 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 Pretreatment 3 step as per the manual Part 1
    • There is a protease digestion step for tissue permeabilization. You need to ensure the temperature is maintained at 40 deg C during the protease digestion step (refer to the user manual)
    • HybEZ is a must have instrument required for RNAscope hybridization as it maintains optimum humidity and temperature (@ 40Deg) 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 RNAscope 2.0 Brown assay.
    • Ecomount Mounting media is required for RNAscope 2.0 Red assay. No other mounting media should be used.
    • Counter staining: diluting 1:2 Gill’s Hematoxylin is required for counter staining
    • ImmEdge Hydrophobic Barrier Pen (CN 310018) is the only pen that will maintain a hydrophobic barrier throughout the RNA scope procedure. No other barrier pen should be used.
  • 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 color channel (C1, C2 or C3) and one of the target probes must be in the C1 channel. Channel C1 target probes are Ready-To-Use (RTU), while channel C2 and C3 probes are shipped as a 50X concentrated stock. The 50x probes for C2 or C3 must be mixed with a C1 Ready to Use Probe. To set up the assay, we recommend you run 3 slides/sample, starting with one with your target probes using a C1 ready to use probe and mixing a channel C2 &/or C3 target of your choice. If no specific C1 probe is used, then a “Blank Probe – C1” (Cat. no. 300041) can be used in place of a specific target probe. The second slide should be the 3 plex control probe and the third would be a DapB negative control probe. You can select any combination based on your imaging instrument configuration. For example, a higher expression gene can be used on a lower wavelength (i.e. green channel). The color channels for the RNAscope® Multiplex Fluorescent Assay are shown in the following table:

    Always use the 3-plex control probes(Hs/Ms with housekeeping genes), for either a duplex chromogenic or a 3-plex multiplex fluorescent assay. This will help interpretation of your results, along with a DapB

    negative control probe slide.

  • Can I omit my C1 probe from the multiplex/duplex assay? How do I use the blank probe?

    C1 probe can be substituted with C1 blank probe diluent and used with C2 and C3 target probes for multiplex/duplex assay.  Do not use channel C2 or channel C3 probes with RNAscope 2.0 Brown or 2.0 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, you can use the slides within 3 months.  Store in dessicant at room temp to avoid RNAses
    • After deparaffinization of tissue preparation, it’s required to use slides within one day
  • Tissue fixation questions: How should my tissue be fixed? Can I use 4%PFA instead of ready to use

    ACD recommends tissue fixation, in accordance to standard clinical research guidelines with fixing samples in FRESH 10% NBF for 16–32hrs at Room Temperature. 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. Yes, freshly prepared 4% paraformaldehyde (PFA) can be used for RNAscope samples. However, fixation should be for 24 hours at room temperature. For optimum results, we highly recommend using the 10% NBF tissue fixation methodology. Please refer to the RNAscope user manual, and note for FFPE tissues, ACD has also provided a Tissue Fixation Pretreatment Guide Chart for guideline, select your tissue type and follow the recommendation on pretreatment conditions. (http://www.acdbio.com/technical-support/downloads/tissue-requirements-troubleshooting-tips/).

    NOTE: Cut embedded tissue into 5 +/- 1 μm sections using a microtome.

RNAscope Transition from 2.0 to 2.5

  • Why should I switch to RNAscope® 2.5 HD?

    The new 2.5 assay will be easier to use and more reproducible resulting in improved product performance. Manual chromogenic Brown/Red kits will require fewer steps for tissue optimization, and detection reagents no longer need to be pre-warmed.

  • When will RNAscope® 2.5 HD kits be available?

    The product will be ready to ship on October 14, 2015, (in the U.S. & E.U.). Contact your ACD account manager to order. Direct online orders will be available in the U.S. by October 22, 2015.

    CLICK HERE FOR 2.5 PNS

  • Will RNAscope® 2.0 HD be discontinued?

    As of October 14, 2015 we will be ready to ship RNAscope® 2.5 kits. If you have an RNAscope® 2.0 HD order in process and would like to switch, contact your ACD account manager. Note: RNAscope® 2.0 HD will officially be discontinued on June 30, 2016 and support will continue to June 1, 2017.

  • I am in the middle of a project, can I still order RNAscope® 2.0 HD kits?

    Yes, RNAscope® 2.0 HD will be in stock for a limited time. Please contact your sales representative for more information.

  • Can I still use my existing probes with RNAscope® 2.5 HD Brown/Red Reagent or Detection kits?

    Yes, you can still use your existing probes. Please refer to the expiration date on the label.

  • Which pretreatment kits should I use with RNAscope® 2.5 HD Brown/Red Reagent or Detection kits?

    For optimal performance with RNAscope® 2.5 HD detection reagents, use the following pretreatment kits: RNAscope® Hydrogen Peroxide, Target Retrieval Reagents, and Protease Plus, Protease III & Protease IV Reagents, as needed.

    Click Here for more information

  • I still have left over Pretreat 1, Pretreat 2, and Pretreat 3. Can I use them with my 2.5 kits?

    No. RNAscope® 2.5 HD has been optimized with RNAscope® Hydrogen Peroxide, Target Retrieval Reagents, and Protease Plus, Protease III & Protease IV.

  • Which pretreatment kits should I use with RNAscope® 2-plex (Red/Green) or Fluorescent Multiplex Detection and Reagent kits?

    For optimal results use the new pretreatment reagents: RNAscope® Hydrogen Peroxide, Target Retrieval Reagents, and Protease Plus, Protease III & Protease IV, depending on your tissue type (FFPE, fixed or fresh frozen, or cultured cells).

Probe Design

  • What is the minimum sequence length of the target region required for RNAscope probe pool design?

    300 to 1000 bases. A typical probe design includes 20 oligo pairs. Each oligo has two hybridizing regions and we refer 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 in to our design strategy, so out of the 20 ZZ oligo pairs only 3 oligo pairs need to bind to generate visible signal. So if your target region has less than 1000 unique bases, we can design a probe consisting of as few as 6 oligo pairs spanning 300 bases that should still be effective for detection of relatively highly expressed RNAs.

     

  • 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 C1 Ready-to-Use probe can be used in each manual assay format – single-plex for chromogenic (2.0), 2-plex chromogenic and fluorescence multiplex. With the duplex and the multiplex you would need a channel 2 or 3 probe for the multiplexing. 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 when they are stored as recommended at 4deg C.

     

  • What is ACD’s process for probe quality control, is each probe tested?

    ACD probe design algorithm is validated to select oligos with compatible melting temperature for optimal hybridization at RNAscope assay condition 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 We do 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: we need >90% homology 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). We will get back to you with the requested information.

     

Tissue Preparation

  • Tissue fixation questions: How should my tissue be fixed? Can I use 4%PFA instead of ready to use

    ACD recommends tissue fixation, in accordance to standard clinical research guidelines with fixing samples in FRESH 10% NBF for 16–32hrs at Room Temperature. 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. Yes, freshly prepared 4% paraformaldehyde (PFA) can be used for RNAscope samples. However, fixation should be for 24 hours at room temperature. For optimum results, we highly recommend using the 10% NBF tissue fixation methodology. Please refer to the RNAscope user manual, and note for FFPE tissues, ACD has also provided a Tissue Fixation Pretreatment Guide Chart for guideline, select your tissue type and follow the recommendation on pretreatment conditions. (http://www.acdbio.com/technical-support/downloads/tissue-requirements-troubleshooting-tips/).

    NOTE: Cut embedded tissue into 5 +/- 1 μm sections using a microtome.

  • What’s the impact of under-fixed or over-fixed FFPE tissue specimens on RNAscope® FFPE Assay?

    Under-fixed 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?

    We understand in many situations you don’t have information on how the tissue was prepared, in such cases, simple optimization steps need to be followed. Or if you know that your tissues are prepared differently from our recommendations, you may need to vary one or both of the pretreatment steps (P2 and P3 referring to the boiling and the protease digestion times). Always run your tissues with ACD control slides (Hs or Ms) using PPIB (positive) and DapB(negative) control probe. Observe the staining pattern, 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, which simply implies identifying the optimum tissue pretreatment conditions by varying P2 or P3 or both. For over-fixed tissues (tissue morphology looks good but staining is weak) to increase probe accessibility you will need to increase P2 or boiling times. For under-fixed tissues (where tissue appears faded, with loss of cell borders) decrease the boiling as it indicates loss of RNA due to protease over-digestion.

  • Can RNAscope assays work with different tissue types?

    RNAscope manual assay can be used with FFPE, fresh-frozen, fixed-frozen and cultured cells. RNAscope automated assays are primarily supported with the FFPE tissue.

  • 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. Please refer to RNAscope® Sample Preparation and Pretreatment Guide for FFPE: http://www.acdbio.com/technical-support/downloads/technical-doc/rnascope-ffpe-tissue-specimen-prep/

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, 2x.
    • 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. For the manual assay, a critical piece of benchtop equipment for routine success with RNAscope is ACD’s HybEZ System (http://www.acdbio.com/products/hybez). 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 XT or ULTRA platform or the Leica Biosystems BOND RX, then we 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 Pretreatment 3 step as per the manual Part 1
    • There is a protease digestion step for tissue permeabilization. You need to ensure the temperature is maintained at 40 deg C during the protease digestion step (refer to the user manual)
    • HybEZ is a must have instrument required for RNAscope hybridization as it maintains optimum humidity and temperature (@ 40Deg) 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 RNAscope 2.0 Brown assay.
    • Ecomount Mounting media is required for RNAscope 2.0 Red assay. No other mounting media should be used.
    • Counter staining: diluting 1:2 Gill’s Hematoxylin is required for counter staining
    • ImmEdge Hydrophobic Barrier Pen (CN 310018) is the only pen that will maintain a hydrophobic barrier throughout the RNA scope procedure. No other barrier pen should be used.
  • 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 color channel (C1, C2 or C3) and one of the target probes must be in the C1 channel. Channel C1 target probes are Ready-To-Use (RTU), while channel C2 and C3 probes are shipped as a 50X concentrated stock. The 50x probes for C2 or C3 must be mixed with a C1 Ready to Use Probe. To set up the assay, we recommend you run 3 slides/sample, starting with one with your target probes using a C1 ready to use probe and mixing a channel C2 &/or C3 target of your choice. If no specific C1 probe is used, then a “Blank Probe – C1” (Cat. no. 300041) can be used in place of a specific target probe. The second slide should be the 3 plex control probe and the third would be a DapB negative control probe. You can select any combination based on your imaging instrument configuration. For example, a higher expression gene can be used on a lower wavelength (i.e. green channel). The color channels for the RNAscope® Multiplex Fluorescent Assay are shown in the following table:

    Always use the 3-plex control probes(Hs/Ms with housekeeping genes), for either a duplex chromogenic or a 3-plex multiplex fluorescent assay. This will help interpretation of your results, along with a DapB

    negative control probe slide.

  • Can I omit my C1 probe from the multiplex/duplex assay? How do I use the blank probe?

    C1 probe can be substituted with C1 blank probe diluent and used with C2 and C3 target probes for multiplex/duplex assay.  Do not use channel C2 or channel C3 probes with RNAscope 2.0 Brown or 2.0 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, you can use the slides within 3 months.  Store in dessicant at room temp to avoid RNAses
    • After deparaffinization of tissue preparation, it’s required to use slides within one day
  • 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 we have extensively tested and validated. Other incubators/hybridization stations do 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.

RNAscope Assay Visualization and Interpretation

  • Can my fluorescent microscope work with RNAscope assay? For fluorescent multiplex assays, what are the probe channels and what are the excitation emission wavelengths of the fluorophores?

    The RNAscope® Fluorescent kits are primarily targeted for fresh frozen and cultured cells. This is mainly due to imaging and analysis challenges with interference from tissue autofluorescence. We recommend the Leica DM series or equivalent or Zeiss Axio Imager. You can run the RNAscope® Fluorescent kit on solid tumor FFPE tissues if you have access to a multi-spectral imaging system, such as Nuance FX (Nuance) and Vectra from PerkinElmer. For specific excitation wavelength of each probe channel please refer to page 7 of our RNAscope® Fluorescent Multiplex Kit User Manual PART 2 which can be found here http://www.acdbio.com/technical-support/downloads/technical-doc/rnascope-mui-fluorescent-user-manual/

  • How do I quantify RNAscope data/staining?

    RNAscope detects individual RNA molecules, which appear as distinct dots, you can use semi-quantitative RNAscope scoring e.g., 1+ to 4+ based on number of dots per cell to interpret staining patterns. We also provide for chromogenic single-plex staining RNAscope® SpotStudio™ v1.0 Software, advanced image analysis solution designed to quantify RNA molecules (“dots”) on a cell-by-cell basis in stained fixed tissue.
    Please visit our website for more information: http://www.acdbio.com/products/spotstudio-software

  • How do I interpret RNAscope data/staining?

    We always recommend running 3 slides per sample, your target marker a positive & a negative control probe. Retrospectively collected tissue specimens may not be prepared according to the Specimen Preparation Guideline. Their RNA quality is also unknown. The UBC (POLR2A for RNAscope® 2.0) positive control will help determine whether the RNA in the tissue specimen is of sufficient quality for detecting your RNA biomarker. The bacterial dapB negative control will help determine whether the tissue specimen is appropriately prepared. Only when the UBC positive control has a score of 3+/4+ (POLR2A 1+) and the DapB negative control has a score of 0/1+, you can confidently make a call on the expression of your target RNA in the tissue specimen

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