Question: How does slide storage or running slides through two Xenium Analyzer runs impact data?
Answer: Here we demonstrate in the following experiments that long-term storage for fresh frozen mouse brain (FF mBrain) and FFPE human breast (hBreast) samples does not significantly impact sensitivity for Xenium v1. Furthermore, running Xenium v1 or Xenium Prime slides twice through the Xenium Analyzer does not significantly change data output for both Gene Expression and Cell Segmentation Staining data.
Xenium v1
Slide Storage - mBrain and hBreast
A time course slide storage experiment was set up with slides containing either FF mBrain or FFPE hBreast sections. A single sample block was used with three sections per slide with serial sections onto slide 1, slide 2, slide 3, etc. in the top position before moving on to the middle position of slide 1, slide 2, slide 3. Therefore, while sections within a single slide are from the same block, they are not serial sections.
Following completion of Xenium In Situ Gene Expression with Cell Segmentation staining, slides were stored either short-term (4°C) or long-term (-20°C). The following time points were tested for each tissue type:
- 1 day in PBS-T
- 1 week in PBS-T then transferred to cryoprotectant for 1 week
- 2 weeks in cryoprotectant
- 4 months in cryoprotectant
Three tissue sections were tested for each condition:
Figure 1. Median transcripts per cell for each storage condition. Each data point represents data from one section and error bars indicate standard error. The median transcripts per cell was not significantly different when comparing storage conditions to the one day PBS-T control*. We estimate that the primary source of variation in transcripts per cell within a condition is due to biological variation of different sections within the block. In general, more internal sections have higher transcript numbers due to less RNA degradation.
*In poor quality tissue samples, median transcript per cell may decline after extended storage in cryoprotectant (data not shown).
Consecutive Xenium Analyzer Runs - FF human tissue array
If slides are kept hydrated and rerun on the instrument (e.g. consecutive runs on the same slide), we expect minimal impact to both gene expression data and cell segmentation outputs. Some variability may be expected depending on the tissue type, input sample quality, and on-slide tissue processing.
Tissue tested: A FF human tissue array containing eight different tissue sections was processed through the Xenium In Situ Gene Expression with Cell Segmentation protocol (CG000749) using the Human Multi-Tissue and Cancer Panel. From left to right: Breast, Brain, Colon, Lung, Pancreas, Liver, Skin, Kidney.
Slide 1 (Rerun) was subjected to two back-to-back instrument runs to generate Xenium Gene Expression with Cell Segmentation data. Slide 2 (Control) slide was a serial section of the tissue array that went through a single instrument run.
| Run 1 | Run 2 | |
| Slide 1: Rerun | Xenium In Situ Gene Expression with Cell Segmentation | Xenium In Situ Gene Expression with Cell Segmentation |
| Slide 2: Control | Xenium In Situ Gene Expression with Cell Segmentation | none |
To assess impact to assay performance, median transcripts were compared between Rerun (Slide 1) and Control (Slide 2) for each tissue type. The median transcripts per cell metric was found to be comparable in both conditions for all tissue types surveyed:
Cell segmentation performance was also found to be comparable:
A small amount of variance was observed in human brain samples.
Furthermore, rerunning slides did not significantly increase background as demonstrated by low negative control probes in each condition.
Xenium Prime
A Xenium Prime slide was subjected to two seven day back-to-back instrument runs to generate Xenium Gene Expression. Data was found to be comparable for multiple tissues tested.
Conclusions
Taken together, we demonstrate in pilot studies shown here that slide storage as well as slide rerunning is low-risk for Xenium v1. However, the ability to obtain comparable data from the same slide after storage or in consecutive instrument runs depends on the initial sample quality, disease state, and on-slide tissue processing.
In the event of a Xenium Analyzer run failure, please reach out to support@10xgenomics.com for further guidance on slide storage and potential impact. Note: if slides are found dehydrated on the instrument, assay performance will be compromised in all cases, and rerun is not recommended.
Product: Xenium In Situ Gene Expression, Multimodal Cell Segmentation, Xenium Prime In Situ Gene Expression