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Methods for the extraction and RNA profiling of exosomes

World Journal of Methodology 2013年第1期3卷 11-18页

作者： Emily Zeringer Mu Li tim barta Jeoffrey Schageman Ketil Winther Pedersen Axl Neurauter Susan Magdaleno Robert Setterquist Alexander V Vlassov Life Technologies

AIM:To develop protocols for isolation of exosomes and characterization of their RNA ***:Exosomes were extracted from He La cell culture media and human blood serum using the Total exosome isolation(from cell culture media)reagent,and Total exosome isolation(from serum)reagent *** and purity of the exosomes was confirmed by Nanosight?analysis,electron microscopy,and Western blots for CD63 *** RNA cargo was recovered with the Total exosome RNA and protein isolation ***,RNA was profiled using Bioanalyzer and quantitative reverse transcriptionpolymerase chain reaction(q RT-PCR)***:Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum,with subsequent isolation and analysis of RNA residing within these *** isolation procedure is completed in a fraction of the time,compared to the current standard protocols utilizing ultracentrifugation,and allows to recover fully intact exosomes in higher *** were found tocontain a very diverse RNA cargo,primarily short sequences 20-200 nt(such as mi RNA and fragments of m RNA),however longer RNA species were detected as well,including full-length 18S and 28S r ***:We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes,followed by isolation of RNA and its analysis by q RT-PCR and other techniques.

关键词： Exosomes Microvesicles Cell culture media Serum RNA Quantitative reverse transcription-polymerase chain reaction Sequencing

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