Extracellular Particles Isolated from Leftover Discarded Formalin-Fixed Tissues Exhibit Atypical Extracellular RNA Profiles.

This study reports atypical findings indicating that extracellular particles (EPs) isolated from leftover discarded formalin-fixed tissues (FFTs) contain atypical RNA profiles that may be highly fragmented. Using particle purification liquid chromatography (PPLC), we isolated EPs from five tissue types that had been immersed in 10% neutral buffered formalin, stored at room temperature, and collected at the point of discard. Tissues were de-identified and maintained at room temperature until EP separation and exRNA purification. Purified EPs were stored at -80 °C before physicochemical characterization and small RNA sequencing. PPLC separation profiles were broadly consistent with expected EP signatures, although subtle shifts in physicochemical properties were observed. exRNA was successfully recovered from all samples; however, RNA-Seq analysis showed that most reads were unclassified, while others mapped to microbial taxa rather than host-derived transcripts. These results suggest that EP-associated RNA in FFT may be substantially degraded and that microbial particles or nucleic acids were present in the tissues despite formalin fixation. Microbial viability was not assessed, as this was outside the scope of the study. Nonetheless, several explanations are plausible, including RNA fragmentation due to prolonged formalin exposure, microbial contamination during pre-discard storage, or inadequate or ineffective fixation that permitted microbial persistence or enrichment. Collectively, these findings indicate that leftover discarded tissues that were not flash-frozen or properly stored may be unsuitable-or at minimum require caution-when used as a source of EPs or EP-associated RNA. Investigators should consider RNA degradation/fragmentation, microbial confounders, and fixation-related artifacts when interpreting EP or exRNA data derived from FFT.