This protocol describes how to collect small-scale plasma samples in order to detect, identify and quantify extracellular RNA.
Method Article
Plasma Collection Procedure (Small Scale) for the analysis of extracellular RNA
https://doi.org/10.1038/protex.2015.098
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This protocol describes how to collect small-scale plasma samples in order to detect, identify and quantify extracellular RNA.
Extracellular RNAs (exRNAs) have been identified in every biofluid that has been tested. They have been found in extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. exRNAs are interesting because they may serve as signalling molecules between cells, they have the potential to serve as biomarkers for prediction and diagnosis of disease, and exRNAs or the extracellular particles that carry them might be used for therapeutic purposes.
The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium (ERCC) is a group of laboratories funded by the U.S. National Institutes of Health to develop robust and standardized methods for collecting and processing of biofluids, separating different types of exRNA-containing particles and isolating and analyzing exRNAs. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.
A full list of the protocols developed during this effort is available at the exRNA Portal, the ERCC's website ( "http://exrna.org/resources/protocols/":http://exrna.org/resources/protocols/ ). This protocol for collecting plasma is one of the biofluid collection and processing methods compared in "the associated publication":http://www.journalofextracellularvesicles.net/index.php/jev/article/view/26533.
Butterfly Blood Collection Set, 21g: Vendor: Becton-Dickson, Catalog # 367287
Vacutainer Holder: Vendor: Becton-Dickson, Catalog # 364815
K2EDTA tube, 10mL: Vendor: Becton-Dickson, Catalog # 366643
Latex-Free Tourniquet: Vendor: Medicus Health, Catalog # 5343M1
FalconTM 15mL Conical Centrifuge Tubes, polypropylene: Vendor: Fisher, Catalog # 14-959-70C
Sterile Microcentrifuge Tubes with Screw Caps: Vendor: Fisher, Catalog # 2681375
See figure in Figures section.Temperatures under 20°C will cause platelet activation ( "PMID 10779050":http://www.ncbi.nlm.nih.gov/pubmed/10779050 ).
As an additional step to remove residual cells, the plasma may be passed through a 0.8 μm cellulose acetate filter. This step should be performed slowly to avoid shear damage to cells or extracellular vesicles. This step may deplete the plasma of larger vesicles, such as oncosomes.
The authors declare no competing financial interests.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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