A Novel Cell Membrane-Associated RNA Extraction Method and Its Application in the Discovery of Breast Cancer Markers

Cellmembrane-associated RNA (mem-RNA) has been demonstrated tobe cell-specific and disease-related and are considered as potentialbiomarkers for disease diagnostics, drug delivery, and cell screening.However, there is still a lack of methods specifically designed toextract mem-RNA from cells, limiting the discovery and applicationsof mem-RNA. In this study, we propose the first all-in-one solutionfor high-purity mem-RNA isolation based on two types of magnetic nanoparticles,named MREMB (Membrane-associated RNA Extraction based on MagneticBeads), which achieved ten times enrichment of cell membrane componentsand over 90% recovery rate of RNA extraction. To demonstrate MREMB'spotential in clinical research, we extracted and sequenced mem-RNAof typical breast cancer MCF-7, MDA-MB-231, and SKBR-3 cell linesand non-neoplastic breast epithelial cell MCF-10A. Compared to totalRNA, sequencing results revealed that membrane/secreted protein-encodingmRNAs and long noncoding RNAs (lncRNAs) were enriched in the mem-RNA,some of which were significantly overexpressed in the three cancercell lines, including extracellular matrix-related genes COL5A1 andlncRNA TALAM1. The results indicated that MREMB could enrich membrane/secretedprotein-coding RNA and amplify the expression differences of relatedRNAs between cancer and non-neoplastic cells, promising for cancerbiomarker discovery.