RESUMEN
Extracellular vesicles (EVs) have been recognized as a rich material for the analysis of DNA, RNA, and protein biomarkers. A remaining challenge for the deployment of EV-based diagnostic and prognostic assays in liquid biopsy testing is the development of an EV isolation method that is amenable to a clinical diagnostic lab setting and is compatible with multiple types of biomarker analyses. We have previously designed a synthetic peptide, known as Vn96 (ME kit), which efficiently isolates EVs from multiple biofluids in a short timeframe without the use of specialized lab equipment. Moreover, it has recently been shown that Vn96 also facilitates the co-isolation of cell-free DNA (cfDNA) along with EVs. Herein we describe an optimized method for Vn96 affinity-based EV and cfDNA isolation from plasma samples and have developed a multiparametric extraction protocol for the sequential isolation of DNA, RNA, and protein from the same plasma EV and cfDNA sample. We are able to isolate sufficient material by the multiparametric extraction protocol for use in downstream analyses, including ddPCR (DNA) and 'omic profiling by both small RNA sequencing (RNA) and mass spectrometry (protein), from a minimum volume (4 mL) of plasma. This multiparametric extraction protocol should improve the ability to analyse multiple biomarker materials (DNA, RNA and protein) from the same limited starting material, which may improve the sensitivity and specificity of liquid biopsy tests that exploit EV-based and cfDNA biomarkers for disease detection and monitoring.
Asunto(s)
Ácidos Nucleicos Libres de Células , Vesículas Extracelulares , Biomarcadores de Tumor , Humanos , Biopsia Líquida , ARNRESUMEN
Soxhlet (SE), microwave-assisted (MAE) and ultrasound-assisted (UAE) extraction were compared using ten extraction solvents for their efficiency to extract phenolic and flavonoid antioxidants from Eastern Canada propolis. Extracts were compared for total phenolic (TPC) and total flavonoid (TFC) content, and radical scavenging activities. Anti-inflammatory activity through inhibition of 5-lipoxygenase (5-LO) products biosynthesis in HEK293 cells was also evaluated. The results showed that SE extracts using polar solvents had the highest TPC and TFC. Extracts obtained with ethanol, methanol and acetone were effective free radical scavengers, and showed 5-LO inhibition similar to zileuton. UAE was an effective extraction method since the extracts obtained were comparable to those using SE and the MAE while being done at room temperature. With UAE, extracts of less polar solvents showed similar free radical scavenging and 5-LO inhibition to extracts of much more polar solvents such as methanol or ethanol. Reversed-phase liquid chromatography tandem mass spectrometry confirmed the presence of 21 natural compounds in the propolis extracts based on the comparison of intact mass, chromatographic retention time and fragmentation patterns derived from commercial analytical standards. The current study is the first of its kind to concurrently investigate solvent polarity as well as extraction techniques of propolis.