Question: Can an FFPE tissue slide be used as input for the Isolation of Cells from FFPE Tissue Sections Demonstrated Protocol (CG000632)?
Answer: Based on limited testing (using one human endocervical cancer and one healthy human lymph node block), we have successfully generated Single Cell Gene Expression Flex libraries from dissociated tissue isolated from twenty tissue slides (5 μm FFPE tissue sections on glass slides; ~100 µm of total tissue). For brevity, this method will be called “Slide Scraping” in this article. To perform the Slide Scraping protocol, we Deparaffinized and Rehydrated tissue (while still on slides) by performing the following:
- Two Xylene washes for 10 min
- Two 100% Ethanol washes for 3 min
- Two 95% Ethanol washes for 3 min
- One 70% Ethanol wash for 3 min
- One Nuclease-free Water wash for 20 sec
- Once 1X PBS wash for 20 sec
After Rehydration, the tissues sections were scraped off the slide at a 45 degree angle with a razor blade (Stanley High Carbon Steel Single Edge Razor Blade 1-½”) and transferred to a 1.5 mL Eppendorf tube. To isolate cells, we followed the Pestle Dissociation of FFPE Tissue Protocol starting at Step 2a.
We found that the Slide Scraping method, compared to the OctoDissociator method outlined in the scFFPE Demonstrated Protocol (using 2x 25 µm freshly sectioned curls), generated comparable cells counts when using approximately twice the amount of sample input (Table 1). Data generated from cells dissociated from lymph node using the Slide Scraping method showed a modest decline in cell yields (as determined by cell counts and the “Estimated Number of Cells” metric in the Web Summary File). Surprisingly, assay sensitivity (as indicated by the “Median UMIs per Cell” and “Median Genes per Cell” metrics in the Web Summary File), usable data (determined by “Confidently Mapped Reads in Cells” metric in the Web Summary File), and cell type annotations (not shown) were similar between these two methods. Data generated from freshly isolated FFPE curls and tissue slides with endocervical cancer had similar data quality.
Table 1. Summary of Gene Expression Flex data generated from samples isolated from freshly sectioned tissue curls (Tissue Curls) and scraping sections from slides (Slide Scraping).
|Tissue Type||Isolated Via||Cell Count||Estimated Number of Cells||Median UMIs per Cell||Median Genes per Cell||Fraction Reads in Cells|
|Endocervical Cancer||Tissue Curls||2.34E6||5,200||950||600||72%|
|Healthy Lymph Node||Tissue Curls||4.68E6||7,100||1,400||1,050||82%|
Together, these data illustrate that it is possible to generate Gene Expression Flex data from sections on slides. Unfortunately, given the amount of sample needed (approximately double compared to OctoDissociator method (~100 µm vs ~50 µm for human samples)) for input into the Slide Scraping protocol, we would recommend for optimal assay performance to utilize freshly isolated FFPE curls.
Please see the “Cell Yields from FFPE Tissue Sections” table in the scFFPE Demonstrated Protocol when optimizing a new sample type. Keep in mind, if using tissue slides, double the amount of tissue listed in the table may be required to ensure the minimum sample input requirements are met (200,000 cells or 400,000 nuclei per Hybridization).
Products: Fixed RNA Profiling, Single Cell Gene Expression Flex