Question: Is Xenium compatible with tissues expressing a reporter protein such as GFP?
Answer: Xenium is compatible with tissue sections that are expressing reporter proteins such as GFP and mCherry. This said, fluorophores are particularly sensitive to any alcohol. The fluorescent reporter will likely be lost as part of Xenium sample preparation and the endogenous fluorescence will not be retained. Therefore is not likely to be a source of endogenous autofluorescence or interfere with data acquisition on the Xenium Analyzer. However, if reporter readout is desired, the reporter protein structure is likely maintained and may be detected with anti-GFP Abs.
Considerations for protein-based detection via IF (unsupported and low risk):
Antibody-based detection methods (immunofluorescence labeling, IF immunohistochemistry (IHC) staining) may enable reporter gene detection paired with Xenium. Because we have not tested this in-house, we cannot provide detailed guidance.
Here are a few recommendations for the proposed experiment:
- The sample would be processed similarly to any other FFPE or fresh frozen sample input. After the Xenium Analyzer run, the sample would have the autofluorescence quenching solution removed as described in Xenium In Situ Gene Expression - Post-Xenium Analyzer H&E Staining (CG000613)(step 1.1). Instead of proceeding to H&E staining after quencher removal, one would proceed to IF instead.
- More detailed methods can be found in “High resolution mapping of the breast cancer tumor microenvironment using integrated single cell, spatial and in situ analysis of FFPE tissue”. This paper details how one might perform IF staining on a breast cancer FFPE tissue assayed with Xenium.
- We do not recommend IF prior to Xenium Sample Preparation as this may compromise assay performance. In summary, protein-based detection of reporter genes may be theoretically possible, but untested.
Xenium-based reporter gene-based, RNA detection (unsupported and high risk):
There is a finite limit to the total transcripts that can be optically resolved in any particular cell. ‘Optical crowding’ is the phenomenon by which the recognition of certain genes is impaired by the presence of other genes. This is most commonly induced by the inclusion of very highly expressed genes in a gene panel. The signal of the highly expressed gene or genes exceeds the system's detection limit and drowns out genes with lower expression, leading to a decreased quality scores for genes readouts.
Reported genes, such as GFP are often very highly expressed. Extremely highly expressed genes can result in reduced overall sensitivity due to crowding. The degree to which GFP signal might obscure the detection of other genes is likely to be very sample-specific. The level of GFP (or other reporter gene) expression and also the number of cells containing the reporter gene will impact assay performance. If the reporter is low to moderate expression or only expressed in a few cells, it may be possible to design add-on probes to this reporter.
Because reporter gene expression is very sample-dependent and also the risk of including reporter genes is high, inclusion of GFP, mCherry, etc. in add-on panels is not recommended.
Considerations for custom gene inclusion can be found in the Species Standalone Custom and Advanced Custom Panel Design for Xenium In Situ Tech Note.
Products: Xenium In Situ Gene Expression