Formalin-fixed, paraffin-embedded (FFPE) tissue samples are widely used in clinical and research settings to preserve biological specimens. However, one big challenge scientists face when working with FFPE tissues is extracting usable genetic material—specifically intact nuclei—for detailed gene expression analysis. The problem lies in the harsh preservation process, which often damages the RNA inside cells, making high-quality transcriptomic analysis difficult.

Researchers from Southeast University have developed a new method called cryogenic enzymatic dissociation (CED) to address this issue. Their approach rapidly and gently breaks down the tissue to extract nuclei without further damaging the RNA inside. When combined with a single-nucleus RNA sequencing technique called snRandom-seq, the team dubbed their new method “snCED-seq.”

snCED-seq for post-fixed tissues overview

The workflow of snCED-seq for post-fixed tissues includes single nuclei isolation by CED and HED method with the snRandom-seq method used in this study. The steps from nucleus extraction to targeted sequencing are shown. In contrast to HED, the nuclei prepared with CED were morphologically intact without leakage of RNA molecular.

What makes snCED-seq so impressive? Compared to older techniques, it gives researchers about ten times more nuclei per sample, takes less time to perform, and keeps the RNA in better shape. This means scientists can detect more genes per cell, with less background noise from unwanted cellular components like mitochondrial or ribosomal RNA.

To test how useful this is in real-world samples, the team applied snCED-seq to both mouse and human FFPE tissues. In mouse brain samples, they could distinguish between specific cell types involved in Alzheimer’s disease, including two different types of astrocytes (a type of support cell in the brain), microglia (immune cells), and oligodendrocytes (cells that insulate neurons). They also showed that it works on very thin slices of human lung tissue, identifying major cell types with high accuracy.

The results are promising: snCED-seq opens the door to exploring archived clinical tissue samples with far greater depth, offering insights into disease at the cellular level that were previously out of reach. With more reliable data and higher sensitivity, RNA sequencing of FFPE samples could now become a standard part of many biomedical research studies.