To preserve RNA in fresh tissue for spatial analysis, prepare the assay-specific freezing setup before collection, minimize the time tissue remains unfrozen, then keep it frozen through embedding, storage, sectioning, and quality control. Use the current preparation protocol for your assay and tissue: freezing methods and RNA acceptance criteria are not universal.
Choose the spatial assay and preparation workflow first
Before collecting tissue, identify the intended assay, its current fresh-frozen tissue preparation handbook, and any local tissue-specific standard operating procedure. Prepare the freezing setup and tools in advance so the specimen can be processed without avoidable delay. Protocols support prompt freezing, but the cited guidance does not establish a universal collection-to-freezing time limit.
Fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) samples use distinct preparation workflows. The 10x Genomics FFPE guide is for FFPE reagent kits; it is not a substitute for the snap-frozen/OCT Visium workflow. Confirm that the tissue-preparation instructions match the assay generation and reagent kit you plan to use.
Freeze freshly collected tissue promptly using the assay’s method
Minimize the interval between collection and freezing. The 10x Genomics Visium Spatial Protocols – Tissue Preparation Guide says freshly obtained tissue must be snap frozen to limit RNA degradation and avoid crystal formation that can damage morphology.
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For the method described in that guide, tissue is frozen using an isopentane bath cooled with liquid nitrogen. The guide cautions against placing tissue directly in liquid nitrogen: the temperature difference can cause surface boiling, air pockets, and uneven freezing, potentially harming tissue morphology. Treat this as guidance for that documented workflow, not a universal freezing recipe; follow the current instructions for your assay and tissue.
Embed in OCT only as directed by the protocol
Optimal Cutting Temperature compound (OCT) is used in these workflows to preserve tissue structure and provide support during cryosectioning. It does not replace rapid freezing or careful cold-chain handling. The Visium guide describes both freezing and embedding together and freezing tissue first, then embedding it. Thin tissue prone to curling may benefit from simultaneous freezing and embedding, but the choice should follow the applicable protocol.
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The 10x Genomics Xenium In Situ for Fresh Frozen Tissues guide likewise describes OCT as an embedding compound for preserving structure and supporting cryosectioning. Use a compatible OCT embedding compound only where the assay instructions specify it.
Keep blocks and sections frozen during storage and RNA sampling
Maintain the frozen chain after embedding. For RNA-quality sampling in the documented CytAssist fresh-frozen workflow, the 10x Genomics guide calls for pre-cooling the tube, cooling block, and forceps. Transfer collected sections into a pre-cooled tube, keep the tube on dry ice, and store the sample at −80°C or proceed to RNA extraction. Prevent sections from melting during handling and transfer.
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These specific sampling and storage directions are tied to that CytAssist workflow. Check the current assay guide for storage, sectioning, and transfer requirements for other platforms or tissue types.
Independent reader supportYour contribution helps us test, update, and keep practical guides available for everyone.Assess RNA quality against the assay’s own acceptance criteria
Assess RNA quality before committing tissue to the spatial assay, following the applicable protocol. In its CytAssist Fresh Frozen Tissue Preparation Guide, 10x Genomics describes RNA isolation and RIN calculation with a BioAnalyzer or TapeStation, and gives RIN ≥ 4 as the proceed criterion for that documented workflow. That value is protocol-specific; it is not an established universal cutoff for all spatial assays, samples, or laboratories.
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Tissue type, freezing, embedding, block handling, and slide processing can all affect performance. 10x Genomics’ tested-tissue guidance can help inform workflow selection, but listed performance is not a guarantee for a particular specimen. Check current documentation and assess blocks before using scarce samples.
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