Question: How does data generated from Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling (Chop/Fix) and Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling (scFFPE) Demonstrated Protocols compare?
Answer: Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling (Chop/Fix) and Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling (scFFPE) Demonstrated Protocols are expected to generate similar quality single cell data when sample input is of high-quality.
Below are cell clustering comparisons generated using freshly procured mouse tissues that were then either fixed and dissociated following the Chop/Fix Demonstrated Protocol or freshly formalin fixed and paraffin embedded and used as input for the scFFPE Demonstrated Protocol. Performance of freshly procured and preserved (either freshly fixed and dissociated or freshly generated FFPE) human samples are expected to be similar to data shown below.
Data quality will likely be variable when comparing freshly fixed samples to older, archived FFPE blocks as FFPE block qualities (age, fixation and embedding methods, tissue type, pre-fixation tissue quality, etc.) can greatly impact assay performance. Therefore, if you have access to fresh tissue or have the ability to control processing methods, we would advise using fresh or frozen tissue and performing the Chop/Fix Demonstrated Protocol instead of generating FFPE blocks to perform the scFFPE Demonstrated Protocol. Fresh or frozen tissue that’s freshly PFA fixed will maintain RNA quality, likely retain higher cellular diversity, and will more likely generate better quality data than previously fixed FFPE samples.
Figure 1. Fixed RNA Profiling t-SNEs with cell type annotations generated utilizing Chop/Fix (left), Pestle (middle) or Octo Dissociator scFFPE (right) protocols for dissociation of matched mouse spleen tissue. UMAPs show similar clustering patterns and cell populations (legend on right).
Figure 2. Fixed RNA Profiling UMAPs with cell type annotations generated utilizing Chop/Fix (left), Pestle (middle) or Octo Dissociator scFFPE (right) protocols for dissociation of matched mouse kidney tissue. UMAPs show similar clustering patterns and cell populations (legend on right).
As displayed in the figures above, both protocols generate similar clustering, suggesting that cell type resolution using either Demonstrated Protocol is maintained in mouse spleen and kidney tissues (Figure 1-2). Interestingly, we observe that there are variable ratios of cell types between freshly fixed (left plots) and FFPE (middle and right plots) samples. For instance, in Figure 2, ureteric epithelium (magenta) and adipocytes (red) are more abundant in FFPE tissue, as compared to freshly fixed kidney tissue. Together, this suggests that preservation methods and subsequent isolation methods may impact the cell types (and subtypes) observed in the Fixed RNA Profiling data.
Figure 3. Fixed RNA Profiling UMAPs with cell type annotations generated utilizing Chop/Fix (left), Pestle (middle) or Octo Dissociator scFFPE (right) protocols for dissociation of matched mouse liver tissue. UMAPs show similar clustering patterns and cell populations (legend on right).
An additional observation that can be made when comparing data generated from cells isolated from Pestle (middle) and Octo Dissociation (right) methods of FFPE samples. In Figure 3, data generated from cells isolated via Octo Dissociation dissociation retained more plasmacytoid dendritic (“pDC”; magenta) and monocyte (black) cells as compared to data generated from cells isolated by the Pestle method.
Altogether, these data illustrate that overall clustering and cell type characterization is similar when using freshly preserved tissues as inputs for either the Chop/Fix or scFFPE Demonstrated Protocols. When reviewing the data closely, there are differences in how effectively subtypes are identified in Fixed RNA Profiling data generated from cells isolated via Chop/Fix and scFFPE Demonstrated Protocols. Given these differences, we do not support nor recommend comparing samples that are preserved differently (i.e., freshly fixed vs FFPE) or isolated utilizing different methods (e.g., Pestle vs Octo Dissociation methods) as batch effects may impact interpretation.
Note: performance of archived samples may differ as compared to freshly procured samples.
Listed below are the documents mentioned in this article:
- Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling Demonstrated Protocol
- Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling Demonstrated Protocol
Products: Fixed RNA Profiling, Single Cell Gene Expression Flex