Question: What are the sample quality recommendations for optimal performance of the Fixed RNA Profiling assay?
Answer: High-quality single cell or nuclei suspensions should be in the Fixed RNA Profiling assay (i.e., Single Cell Gene Expression Flex) for optimal assay performance. Highly viable single cell or nuclei suspensions (>80%) will have the greatest sensitivity and cell recovery. Although there is not a strict cutoff, we do recommend cleaning up dead cells if you have cell viability <80%.
The Fixed RNA Profiling assay is robust with samples at much lower viability, with successful results demonstrated even with low viability samples (50% or lower). Low viability samples may have more variable cell calling and lower sensitivity. It is important to note that samples with lower viabilities may exhibit signs of stress or higher expression of MT genes.
This could be an indicator of:
- Poor sample quality, leading to a high fraction of apoptotic or lysed cells prior to sample fixation.
- The overall biology of the sample, for example tumor biopsies, which may have increased mitochondrial gene expression due to metabolic activity and/or necrosis.
Samples should have minimal debris for best results; debris can have associated RNA that is detected outside cells.
Cell debris/dead cells cleanup methods are compatible with the Fixed RNA Profiling assay. These include the use of appropriate filters and/or other methods (e.g. FACS, Miltenyi Dead Cell Removal). Please note that a considerable % of cells will be lost during the use of the Dead Cell Removal Kit, so we only recommend the use of this kit only if there is a sufficient cell number, to begin with.
Cellular Debris and cell clumps can be removed using 30 µm filters such as Miltenyi Pre-Separation Filters or Sysmex CellTrics Filters. Please also review: What cell strainers are recommended for use with Fixed RNA Profiling assay?
Cell sorting can also be used to remove dead cells and cellular debris or to enrich a specific population of cells prior to sample fixation.
Products: Fixed RNA Profiling Gene Expression, Single Cell Gene Expression Flex