Question: Are cell types or tissues with high levels of RNases compatible with the Fixed RNA Profiling assay?
Answer: We have performed limited testing in-house on RNase-rich tissues (i.e., spleen). These samples performed similarly to other non-RNase-rich tissues in the Fixed RNA Profiling Assay. Fixation of these samples limits the degradation after collection that is often observed due to high levels of RNases and other inhibitory compounds.
Fresh or Frozen Tissue:
Fresh tissue or frozen can be used in the Fixed RNA Profiling assay by following the 10x Demonstrated Protocol for Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling. This is the preferred sample preparation method for fresh or frozen tissue for optimal assay performance. It is important to note that previously established tissue dissociation protocols for fresh tissue that yielded good results for 3’ GEX are also expected to perform similarly in Fixed RNA Profiling, and can be used with this assay.
The fixation of whole tissues for use in the Fixed RNA Profiling assay is not be supported. Tissue samples must first be chopped into smaller pieces before fixation for optimal assay performance. This step is required for uniform fixation and permeabilization. Please also see: Which tissue dissociation protocols are supported for use with the Fixed RNA Profiling assay?
Tissue Dissociation:
Tissue dissociation protocols that have been previously tested for compatibility with the Single Cell Gene Expression assay (i.e., 3’ GEX) are expected to perform similarly in the Fixed RNA Profiling assay. If these isolation methods/protocols yielded good results for 3’ GEX, they are expected to perform similarly in FRP. The addition of RNase inhibitors is recommended during sample preparation of RNase-rich tissue. Supplementing RNase inhibitors into the wash and resuspension buffers may also help preserve your RNA before sample fixation. Up to 1U/ul may be used for single cell assays.
During fixed sample storage and throughout the workflow, Enhancer is added to the resuspension and wash buffers. One of the components of the Enhancer is RNase inhibitors, which help to preserve sample quality throughout the workflow. For RNase-rich tissues, we recommend adding RNAase inhibitor (Protector RNase inhibitor: Sigma, PN-3335399001) to samples resuspended in the post-hybridization resuspension buffer before storage at -20°C or -80°C. We recommend a concentration of 0.2U/ul of RNase Inhibitor. For optimal assay performance, we recommend storing post-fixation and post-hybridization samples at -80°C.
Additional considerations and protocol modifications may be necessary. Please review: Can I use RNase-rich tissue samples for single-cell gene expression or Multiome assays? The same general guidelines outlined in this article apply to the Fixed RNA Profiling Assay.
Products: Fixed RNA Profiling Gene Expression