A73-17 Extracellular Vesicle Isolation and RNA Extraction Methods and their Impact on Diagnostic Assay Optimization

Abstract Rationale Optimization of RNA isolation methods are critical for evaluating small non-coding RNAs such as microRNAs (miRNA) as candidate diagnostic, prognostic and therapeutic biomarkers. Previously, we identified tumor-derived pleural fluid (PF) extracellular vesicle (EV) miRNAs that discriminate between malignant (MPE) and non-malignant pleural effusion (non-MPE) with significant accuracy. To optimize candidate biomarker isolation for downstream clinical assay development, we compared extracellular-miRNA expression in MPE following EV enrichment versus cell-free PF without EV enrichment and compared the effect of subsequent RNA isolation method on target extracellular-miRNA expression. We hypothesize that EV enrichment in PF and subsequent RNA isolation methods yield significant differences in candidate extracellular-miRNA expression. Methods Six adenocarcinoma-induced MPE samples were processed and their supernatants were used for analysis. Cell-free PF RNA extraction was performed via manual organic RNA extraction method (AllPrep® Universal Kit) versus automated magnetic particle RNA extraction method (QIASymphony® RNA Kit) (Figure 1A, 1 and 2) per the manufacturer’s protocol. As a comparator, all 6 MPE cases alternatively underwent EV enrichment (Invitrogen, Total Exosome Isolation Reagent, followed by ultracentrifugation) (Figure 1A, 3) prior to automated magnetic particle RNA extraction (Maxwell® RSC miRNA Plasma and Serum Kit protocol). Each sample was mixed with non-human RNA spike-in (osa-miR.414; ath-miR.159a; cel-miR.248) for RNA normalization prior to extraction and underwent gene amplification of a candidate extracellular-miRNA biomarker (miR.144.3p) via reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). The Comparative CT method for relative quantification of gene expression was used to determine miRNA expression levels and 2−Δct values as well as cycle thresholds were used for the biostatistical analysis. Raw and log transformed values were compared across three methods using the Wilcoxon rank-sum test and pairwise spearman correlation. Results Analysis included 6 MPE cases processed via 3 different methods (Figure 1A). RNA spike-in expression as well as microRNA expression (2−Δct and cycle threshold) values varied among the same samples based on isolation method. Relative RNA spike-in (Figure 1B) and miRNA expression were highest in EV enriched PF samples. Among the groups that did not undergo EV enrichment, relative miRNA expression was the lowest within cell-free PF specimens analyzed with an automated magnetic particle extraction method. Conclusions Cell-free pleural fluid microRNA expression varies significantly based on EV enrichment and subsequent RNA isolation method. RNA extraction following EV enrichment may be superior to RNA extraction without EV enrichment in detecting malignant pleural fluid biomarkers and should be evaluated in larger cohorts. This abstract is funded by: Supported in part by the Vanderbilt CTSA grant UL1TR002243 from NCATS/NIH​