How should I evaluate my FFPE samples prior to using the Evercode WT FFPE kit?
The quality of FFPE samples is variable and is due to a variety of factors. We recommend considering the following metrics to evaluate your FFPE sample quality prior to running an EvercodeTM WT FFPE kit:
1. Fixation
For optimal results, use tissues fixed under the following conditions: 10% Neutral Buffered Formalin (NBF) at a 20:1 fixative-to-tissue ratio, incubated for 16–24 hours at 4 °C with gentle agitation.
2. DV200
DV200 is a metric of what percent of the total RNA in a sample is above 200 bases in length. In general, a higher DV200 score results in higher data quality. The minimum DV200 score we recommend for input into an Evercode WT FFPE kit is 30%.
3. Storage
For more optimal sample quality, we recommend using FFPE samples that have been stored at 4°C or that have been vacuum sealed. There is no absolute age cutoff for using FFPE tissue in snRNA-seq experiments, but performance generally declines with increasing block age due to RNA oxidation and degradation. In general, shorter storage times (e.g., <5 years) are preferred, although successful results can still be obtained from archival FFPE samples that are 5-10+ years old.
4. Imaging - if available/feasible
If imaging is available, review an H&E slide to confirm the region has adequate nucleated cellularity and is not dominated by necrosis, fibrosis, or low-cellularity tissue. Be cautious of areas with high necrosis or hemorrhage. In addition, be cautious of stroma-, adipose-, or fibrosis-heavy regions, which often yield low RNA and less informative data. Features like inflammation, mucin, or calcification can also reduce the number of usable nuclei.