Sample Preparation

Under-fixation of tissue specimens will result in protease over-digestion, which leads to loss of RNA and poor tissue morphology. Over-fixed tissue specimen will result in protease under-digestion, which leads to poor probe accessibility and low signal and signal/background ratio while maintaining excellent tissue morphology.

ACD recommends tissue fixation in accordance with standard clinical research guidelines. FFPE samples should be fixed in FRESH 10% NBF (neutral buffered formalin) for 16 – 32 hrs at RT. NOTE: Do not fix at 4°C. Do not fix for < 16 hrs or > 32 hrs. Delayed fixation can degrade RNA and produce lower signal or no signal. Shorter time or lower temperature will result in under-fixation. For optimal results, ACD highly recommends using the 10% NBF tissue fixation methodology.

ACD recommends maintaining RNAse-free environment while handling and sectioning tissues prior to fixation. Once the samples have been properly fixed, further RNA degradation is not expected to occur.

RNAscope, BaseScope and miRNAscope assays can be used with FFPE tissue, fresh-frozen tissue, fixed-frozen tissue, and cultured cells. ACD has user manuals and technical notes and recommendations for preparing each of these sample types. Note that cultured cell samples are not compatible with RNAscope HiPlex v2 assay.

Yes, RNAscope works just as well on properly fixed and prepared TMAs as it does on individual tissue sections. Because there may be variability from core to core in the TMA, optimization of the pretreatment conditions may be required.

ACD understands that in some situations, you may not have information on how the tissue was prepared or the tissues were prepared differently from our recommendations. In that case, you may need to vary one or both pretreatment steps (target retrieval and/or protease digestion). ACD recommends that tissues are tested along with ACD control slides (Hs or Mm) using positive and negative control probes.