Question: Does the FFPE Demonstrated Protocol produce a suspension of single nuclei, single whole cells or a mixture of both?
Answer: The Sample Preparation from FFPE Tissue Sections Demonstrated Protocol outlines how to isolate nuclei from formaldehyde fixed & paraffin embedded (FFPE) tissue sections for use with the Flex Gene Expression workflow.
To characterize the nuclei or cell-like character of FFPE suspensions prepared with the Sample Preparation from FFPE Tissue Sections Demonstrated Protocol, 5 FFPE human blocks from lung cancer, healthy liver, thymus, breast cancer, and glioblastoma tissues were dissociated. Transmission electron microscopy (TEM) at the Stanford Cell Sciences Imaging Facility (CSIF) was used to confirm that all FFPE tissues yielded suspensions of particles that contained a preserved nuclear envelope (examples in Image 1 and Image 2), but lacked a cytoplasmic membrane as seen in the control PBMC sample (Image 3). Based on the TEM images it was concluded that it would be appropriate to refer to the dissociated FFPE particles as nuclei rather than cells. This finding is in agreement with other publications in the literature characterizing FFPE dissociations including Chung et al. 2022* and Vallejo et al. 2022^.
Image 1: snFFPE dissociations of human liver tissue (left)
Image 2: snFFPE dissociations of human lung cancer tissue (middle)
Image 3: control human PBMCs w/ intact nuclear and cell membranes (right)
* SnFFPE-Seq: towards scalable single nucleus RNA-Seq of formalin-fixed paraffin-embedded (FFPE) tissue. Chung H*, Melnikov A*, McCabe C*, Drokhlyansky E, Van Wittenberghe N, Magee EM, Waldman J, Spira A, Chen F, Mazzilli S, Rozenblatt-Rosen O, Regev A. bioRxiv 2022.
^ snPATHO-seq: unlocking the FFPE archives for single nucleus RNA profiling. Andres F Vallejo, Kate Harvey, Taopeng Wang, Kellie Wise, Lisa M. Butler, Jose Polo, Jasmine Plummer, Alex Swarbrick, Luciano G Martelotto. bioRxix 2022.
Products: Flex Gene Expression