Sample preparation is critical for successful staining of tissue with RNAscope™ ISH methodology. RNAscope™ manual assay can be used with FFPE (formalin-fixed, paraffin-embedded) tissue, cultured cells, fresh-frozen & or 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 available at User manuals to learn about the sample preparation pretreatment methods for the detection assay and to ensure you have the RNAscope™ Pretreatment reagents, before you start the assay.

FFPE Tissue specimens should be blocked into a thickness of 3 - 4 mm and fixed for 24 +/- 8 hours in 10% neutral-buffered formalin at room temperature. The fixed tissues should then be dehydrated in a graded series of ethanol and xylene, followed by infiltration by melted paraffin held at no more than 60°C. Specimens should be analyzed within 3 months of sectioning when stored at room in desiccant temperature (20-25 °C). Slides need to be air dried and baked at 60°C for 1-2 hours prior to the RNAscope™ Assay. FFPE Tissue sections should be cut into sections of 5 +/-1μm. Use Fisher Scientific SuperFrost Plus Slides for all tissue types to avoid tissue loss. Tissue thickness for fixed frozen tissue should be between 7–15 µm and 10–20 µm for fresh frozen tissue.

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. Refer to the troubleshooting guide to learn more about assay optimization.

We recommend you run RNAscope assay with the control slides and test your samples with the ACD control probes. Using control slides will test assay conditions, while using control probes will test the quality of the RNA in your samples. The housekeeping gene PPIB (Cyclophilin B), often used as a reference gene for RT-PCR, can be used as a positive control. The bacterial dapB gene is used as a negative control.
        310045 - RNAscope™ Control Slides--Human Hela Cell Pellet
        310023 - RNAscope™ Control Slides--Mouse 3T3 Cell Pellet

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. 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.

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 prepared according to our recommended protocol by being fixed at Room Temperature (RT) for 16–32 hours in fresh 10% neutral-buffered formalin (NBF). Always start with the pretreatment guideline recommendations as documented in the RNAscope assay user manual. In situations where information on how the tissue was prepared is not available or if tissues were prepared differently than our recommendations, simple optimization steps can help you obtain excellent results with RNAscope assay. Note: Your sample preparation guide includes recommended pretreatment guidelines.