The most common reason for subpar results with the RNAscope™ assay is suboptimal sample preparation. Wherever possible, tissues should be prepared 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.
- Dehydrate in a graded series of ethanol and xylene, followed by infiltration with 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.
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 sample. Optimal conditions are dependent on tissue type, age, and fixation. Simple optimization steps can help obtain quality data.
If sample preparation conditions do not match recommended guidelines or are unknown, qualifying samples prior to performing any experiments is strongly recommended.
Note: Under-fixation will result in significant RNA loss during storage and may result in low signal when performing the RNAscope™ assay.
