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

Area of Science:

• Biochemistry
• Molecular Biology
• Pathology

Background:

• Extracellular particles (EPs) are investigated for their potential as biomarkers.
• Formalin-fixed tissues (FFTs) are a common archival resource, but their suitability for RNA analysis is debated.
• Understanding RNA integrity in EPs from FFTs is crucial for reliable biomarker discovery.

Purpose of the Study:

• To investigate the RNA profiles of extracellular particles (EPs) isolated from discarded formalin-fixed tissues (FFTs).
• To assess the impact of formalin fixation and storage conditions on RNA integrity within EPs.
• To determine the suitability of FFT-derived EPs as a source for extracellular RNA (exRNA) analysis.

Main Methods:

• Isolation of EPs from five types of discarded FFTs using particle purification liquid chromatography (PPLC).
• Physicochemical characterization of isolated EPs.
• Small RNA sequencing (RNA-Seq) to analyze the purified exRNA.
• Analysis of RNA reads for host-derived transcripts versus microbial or unclassified sequences.

Main Results:

• EPs were successfully isolated from FFTs, with PPLC profiles consistent with EPs.
• exRNA was recovered, but RNA-Seq revealed significant fragmentation and a high proportion of unclassified or microbial reads.
• Host-derived transcripts were scarce, suggesting substantial RNA degradation or microbial contamination.

Conclusions:

• EP-associated RNA in FFTs is often highly fragmented and may be confounded by microbial nucleic acids.
• Prolonged formalin exposure, storage conditions, or inadequate fixation may contribute to RNA degradation and microbial presence.
• Leftover discarded FFTs require careful consideration and potential pre-analysis for RNA integrity and microbial contamination before use in EP or exRNA studies.