An Optimised Protocol for Isolation of RNA from Small Sections of Laser-Capture Microdissected FFPE Tissue Amenable for Next-Generation Sequencing

Formalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA-protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with RT-qPCR or next-generation sequencing (NGS) becomes very difficult. To overcome this limitation, we successfully adapted a protocol that uses focused ultrasonication to isolate RNA from our deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach proved to be qualitatively superior compared to the old approach, as Cq values in RT-qPCR were on average 1.9–fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well. Concluding, we present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen.