Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes

作者：Rossella Crescitelli, Cecilia Lässer, Tamás Szabó, Ágnes Kittel, Maria Eldh, Irma Dianzani, Edit I. Buzás, Jan Lötvall · 发表于：Journal of Extracellular Vesicles · 年份：2013 · DOI：10.3402/jev.v2i0.20677 · 被引用次数：1010 · 研究领域：Extracellular vesicles in disease、MicroRNA in disease regulation、Cellular transport and secretion

INTRODUCTION: In recent years, there has been an exponential increase in the number of studies aiming to understand the biology of exosomes, as well as other extracellular vesicles. However, classification of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross-contamination. METHOD: EVS RELEASED FROM THREE DIFFERENT KINDS OF CELL LINES: HMC-1, TF-1 and BV-2 were isolated using two centrifugation-based protocols. In protocol 1, apoptotic bodies were collected at 2,000×g, followed by filtering the supernatant through 0.8 µm pores and pelleting of microvesicles at 12,200×g. In protocol 2, apoptotic bodies and microvesicles were collected together at 16,500×g, followed by filtering of the supernatant through 0.2 µm pores and pelleting of exosomes at 120,000×g. Extracellular vesicles were analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a Bioanalyzer(®). RESULTS: RNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no RNA except for microvesicles collected from TF-1 cell cultures. The different vesicle pellets showed highly different distribution of size, shape and electron density with typical apoptotic body, micro...