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.
RNase inhibitors are recommended for sample preparation of RNase-rich tissue. Supplementing RNase inhibitors into the wash and resuspension buffers may also help preserve your RNA before sample fixation. 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 for tissues rich in RNases to maintain complexity in final gene expression libraries. Please also see: 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