Formalin Fixed Paraffin Embedded (FFPE) Tissue Samples
It is recommended to work in an RNase free environment to avoid degradation of RNA molecules. Always use fresh RNAse free reagents for samples preparation.
Sample Preparation Recommendations
- Immediately following dissection, fix tissue in 10% NBF for 16–32 hrs at room temperature (RT).
- Fixation time will vary depending on tissue type and size.
- Wash sample with 1X PBS.
- Dehydrate sample using a standard ethanol series, followed by xylene.
- Embed sample in paraffin using standard procedures and trim paraffin blocks as needed.
Note: Embedded samples can be stored at RT. For better RNA preservation, we recommend to store samples at 4°C with desiccant over a long period (>1 year).
- Cut embedded tissue into 5 +/– 1 µm sections using a microtome.
- Place paraffin ribbon in a 40–45°C water bath.
- Mount sections on SuperFrost® microscope slides (ThermoFisher Scientific).
- You can place several sections (corresponding to the approximate size of the SecureSeal chamber format you will use) on one slide with few mm distance between them.
- Note: Avoid folds as it results in strong autofluorescent background and influences the outcome of the ISS.
- You can store sections at RT for several months or at 4°C for a longer period. Storage at 4°C ensures RNA preservation for longer periods of time.
If possible, check the RNA quality by Bioanalyzer before sending the samples. We recommend a RIN value > 7 for ISS.
Note: Lower RIN values indicate sub-optimal RNA integrity which will impact the performances of the ISS assay.
Please contact us (at firstname.lastname@example.org) if you are interested in sequencing samples with RIN value below 7.