RNAscope™ ISH Troubleshooting

RNAscope™ ISH Overview

RNAscope technology is an 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 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 Roche DISCOVERY ULTRA or the Leica Biosystems’ BOND RX platforms.

1. Key RNAscope Assay Guidelines
2. RNAscope Recommended Workflow
3. Scoring Guidelines
4. Sample Preparation
5. Optimize the Assay
6. Troubleshooting Staining Patterns
7. Troubleshooting High Background
8. Troubleshooting Other Issues

Key RNAscope Assay Guidelines

Before you get started refer to our Getting Started Page.

Channel C1 target probes are Ready-To-Use (RTU), while channel C2, C3, and C4 probes are shipped as 50X concentrated stocks. To independently detect target RNAs in a 2-plex, 3-plex, or 4-plex assay, the target probes must be in different channels and there must be a C1 or S1 probe in the mixture. RNAscope Probe Diluent (Catalog # 300041) can be used if no C1 probe is included in the assay. All HiPlex target probes are shipped as 50X concentrated stocks so probe mixture must be prepared using hiPlex probe diluent (Catalog # 324301).

Mounting Medium

Use assay specific recommendations as listed in the table below.

RNAscope Recommended Workflow

 If sample preparation conditions do not match recommended guidelines or are unknown, we strongly recommend checking RNA quality and optimizing pretreatment conditions using ACD positive and negative control probes. See Figure 1 for the recommended workflow

1. Run samples along with the control slides provided by ACD (Catalog # 310045 for Human HeLa Cell Pellet and Catalog # 310023 for Mouse 3T3 Cell Pellet).

• The ACD Positive Control Probes include 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 (see Figure 2).

• For RNAscope, successful PPIB staining should generate a score ≥2 and UBC score ≥3 with relatively uniform PPIB/POLR2A/UBC signal throughout the sample. See user manuals for BaseScope or miRNAscope scoring guidelines.
• 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.

4. Depending on staining results, you may need to optimize pretreatment conditions for your samples.

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. For BaseScope™ or miRNAscope™ scoring guidelines, please reference respective assay user manuals found on product pages.

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, or fixed frozen tissues. Please refer to the appropriate sample preparation guides as shown in the User Manual Selection Guide (found in the documents section of each product page) 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 and 4-6 µm for FFPE tissue. Use Fisher Scientific SuperFrost Plus Slides for all tissue types to avoid tissue loss.

Pointers 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.

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. If you intend 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.

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.
• 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.

Troubleshooting Staining Patterns

1. Staining examples for various pretreatment digestion conditions

Note: When optimizing RNAscope, BaseScope, or miRNAscope workflows, please use a combination of Target Retrieval and -Protease or Protease only (fresh frozen tissue).

Figure 4. Possible Morphology Patterns

2. Staining Patterns

 **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.

Troubleshooting High 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. 

Figure 5: Background Troubleshooting Examples

Troubleshooting Other Issues 

Example: