Question: Can I substitute MilliQ water for nuclease-free water in the Xenium workflow?
Answer: Nuclease-free reagents are used in RNA-based assays to prevent the loss of DNA and RNA during molecular biology work. For Xenium, it is important to use nuclease-free reagents (for example water) to prevent RNA (Xenium template) or DNA (Rolling Circle Product, RCP) degradation during the assay workflow or during the instrument run. Degraded RNA or RCPs can lead to decreased sensitivity.
At many steps in both the Xenium In Situ Gene Expression assay and Xenium Analyzer workflow, bottled nuclease-free water (Thermo Fisher Scientific, AM9937) is a required third-party reagent. Alternate brands of bottled nuclease-free water can be substituted, as described in: Can I use alternative third-party reagents for Xenium?
Here we describe considerations for nuclease-free water use.
Assay Workflow
Nuclease-free water cannot be substituted with Milli-Q* water during the assay workflow. The Xenium workflow has been carefully designed to maintain RNA integrity and this reagent swap may compromise assay performance.
Instrument Run
For the Xenium Analyzer instrument run, it is possible to use nuclease-free Milli-Q water instead of bottled nuclease-free bottled water (Thermo Fisher Scientific AM9937) for the buffers in Bottles 1-4. We have tested nuclease-free Milli-Q water filtered by the Biopak® Polisher (Millipore Sigma CDUFBI0A1)** and observed comparable performance between the Biopak system and bottled Thermo Fisher Scientific water. This reagent swap is validated for instrument buffers only.
We do not recommend using non-nuclease-free water for any buffers in the Xenium In Situ Gene Expression Assay workflow or for any Xenium Analyzer reagents as this may result in decreased assay performance.
Best practices for the Milli-Q filtration systems with Biopak® Polisher (for Xenium Analyzer buffer preparation):
- Confirm the Milli-Q water is bacteria-free, protease-free, endotoxin-free, and nuclease-free
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Before each use, perform a systems check to ensure consumables are not expired
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This can be accessed through filtration system UI for the consumables and also through visual inspection of the component as shown below
- Example of expired component:
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*For any step that uses Milli-Q water, this can be substituted with nuclease-free water (not DEPC-treated).
**Other filtration systems have not been tested and are therefore are unsupported as we cannot confirm assay performance.
Here are some additional nuclease free water options that are not validated by R&D, but would be considered low-risk swap for the instrument run only:
- Sigma Aldrich W4502
- G-Biosciences Molecular Grade Water
Products: Xenium In Situ Gene Expression, Xenium Prime In Situ Gene Expression