Question: How can I avoid mRNA degradation during sample preparation and slide processing for Visium for fresh-frozen?
Answer: We have not observed significant mRNA degradation during our Visium workflow. We have found that our fixed tissue is robust, despite all the staining and rinsing steps performed at room temperature. We would recommend following general best practices when working with RNA (decontaminate benches and pipettes with RNase Away before starting, etc.) and check that you’re getting a good signal on the Tissue Optimization slides prior to moving on to the Gene Expression slides. Additional suggestions to avoid degradation include:
- Use 100% Methanol for fixing tissue sections, HPLC grade that has been pre-chilled - using a mix of methanol/PBS in our hands has led to degraded cDNA (shorter fragments)
- Install a new blade during sectioning for each tissue type
- Equilibrate slides in cryostat prior to sectioning tissue onto the capture areas
- Keep slide in cryostat throughout sectioning and do not let warm once a section has been placed onto the slide
- When transporting slides with tissue sections, transport on dry ice to avoid unintentional freeze-thaw cycles
- Use clean beakers (Spray with RNase Away and/or autoclave prior to use)
- Use nuclease-free water for all steps of the protocol (including H&E rinses)
- Use our recommended brands of staining reagents and/or avoid reusing aliquots of the same staining buffer for multiple tissue sections
- Set-up microscope using the Imaging Test Slide to capture the correct areas on the slide before beginning the Visium workflow to avoid leaving the slide out longer at room temperature than necessary during imaging
- Fix, stain, permeabilize, and process samples through to the first stopping point (post cDNA amplification) in the same day
Product: Visium for fresh-frozen