Question: How can I avoid mRNA degradation during the Visium CytAssist Spatial Gene Expression for Fresh Frozen (FF) assay?
Answer: We have not observed significant mRNA degradation during the Visium CytAssist for FF 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 such as decontaminating benches and pipettes with RNase Away before starting. 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.
- Pre-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 avoid reusing aliquots of the same staining dye for multiple tissue sections.
- Set-up microscope before beginning the workflow to avoid leaving the slide at room temperature longer than necessary during imaging.
- Only pause during recommended stopping points with the provided storage conditions.
Product: Visium CytAssist for FF