Author:
García José Miguel,García Vanesa,Peña Cristina,Domínguez Gemma,Silva Javier,Diaz Raquel,Espinosa Pablo,Citores Maria Jesús,Collado Manuel,Bonilla Félix
Abstract
Little is yet known about the origin and protective mechanism of free nucleic acids in plasma. We investigated the possibility of these free nucleic acids being particle associated. Plasma samples from colon cancer patients and cell culture media were subjected to various antibody incubations, ultracentrifugation, and RNA extraction protocols for total RNA, epithelial RNA, and mRNA. Flow cytometry using a Ber-EP4 antibody and confocal laser microscopy after staining with propidium iodide were also performed. mRNA levels of the LISCH7 and SDHA genes were determined in cells and in culture media. Ber-EP4 antibody and polystyrene beads coated with oligo dT sequences were employed. We observed that, after incubation, total RNA and mRNA were always detected after membrane digestion, and that epithelial RNA was detected before this procedure. In ultracentrifugation, mRNA was caught in the supernatant only if a former lysis mediated or in the pellet if there was no previous digestion. Flow cytometry determinations showed that antibody-coated microbeads keep acellular structures bearing epithelial antigens apart. Confocal laser microscopy made 1- to 2-μm-diameter particles perceptible in the vicinity of magnetic polystyrene beads. Relevant differences were observed between mRNA of cells and culture media, as there was a considerable difference in LISCH7 mRNA levels between HT29 and IMR90 cell co-cultures and their culture media. Our results support the view that extracellular RNA found in plasma from cancer patients circulates in association with or is protected in a multiparticle complex, and that an active release mechanism by tumor cells may be a possible origin.
Publisher
Cold Spring Harbor Laboratory
Cited by
80 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献