Question: Can I use fewer than the minimum recommended number of cells/nuclei during probe hybridization with the Fixed RNA Profiling Assay (i.e., Single Cell Gene Expression Flex assay)?
Answer: As some cell loss is expected throughout the probe hybridization and washing, we recommend starting hybridization with sufficient cells for the assay.
- Singleplex: The recommended minimum input is 200,000 cells or 400,000 nuclei per hybridization. In addition, the recommended minimum input for FFPE dissociated suspension is 400,000 cells per hybridization.
- Multiplex: The recommended minimum input is 50,000 cells or 100,000 nuclei per hybridization (i.e., per probe barcode). In addition, the recommended minimum input for FFPE dissociated suspension is 100,000 cells per hybridization (i.e., per probe barcode).
- We recommend starting with 1 x 10^6 cells whenever possible, with a maximum input of 2 x 10^6 cells/nuclei per hybridization.
The recommended minimum number of cells/nuclei was set to ensure assay success as recovery can vary greatly between sample types. For fixation and hybridization, it may be possible to use less than the recommended minimum. However, there is potential risk of not having enough cells/nuclei for the downstream assay. The recommended minimum is not a strict requirement for assay performance, but this guidance ensures that there are enough cells/nuclei to proceed with the workflow.
Additional steps can be taken to improve cell/nuclei yield. Please review: How can I improve my cell/nuclei recovery using the Fixed RNA Profiling Assay?
Products: Fixed RNA Profiling Gene Expression, Single Cell Gene Expression Flex