Question: Is Xenium compatible with tissues expressing a reporter protein such as GFP?
Answer: It is possible to perform the Xenium workflow on tissue sections with reporter proteins. The reporter will be denatured as part of sample preparation and the endogenous fluorescence will not be retained. Measuring the specific reporter gene or genes in the same tissue section as Xenium Gene Expression is officially untested and unsupported. Theoretically, it may be possible, but there are many experimental design considerations for this experiment. The first question is whether the RNA or protein of the reporter target is of interest. Detecting protein may be theoretically possible post Xenium Analyzer run. Conversely, RNA detection as part of an add-on panel is considered high risk and is officially unsupported.
Considerations for protein-based detection (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.
Considerations for Xenium-based GFP RNA detection (unsupported and high risk):
The Xenium technology uses microscopy imaging to detect individual transcripts in cells. However, 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.
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.
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. Furthermore the current Xenium Panel Designer does not have capabilities to include these.
Recommendations for custom gene inclusion can be found in the Xenium Add-on Panel Design Technical Note (CG000643) (coming soon to support webpage):
- Expression level: Neither have too low (<0.1 transcripts per cell) nor too high (>50 transcripts per cell) expression levels. This can be assessed by single cell experiments.
- Only be expressed in certain cells or cell states.
- In summary, we do not recommend RNA-based detection of reporter genes.
Products: Xenium In Situ Gene Expression