In:
Journal of Extracellular Vesicles, Wiley, Vol. 7, No. 1 ( 2018-12)
Kurzfassung:
Sample amount is often a limiting factor for multi‐parametric analyses that encompass at least three areas of ‘‐omics’ research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi‐parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi‐parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein‐extraction reagents. EVs were isolated from 3 mL of cell‐culture supernatant derived from MCF‐10A, MCF‐7 and MDA‐MB‐231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re‐solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris‐HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS‐PAGE. NanoLC‐MS/MS of the trypsin‐digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF‐10A, MCF‐7 and MDA‐MB‐231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol© reagent is the ability to accumulate multi‐parametric data (e.g., RNA and protein profiles) on the same limited EV sample while minimizing sample preparation and processing time.
Materialart:
Online-Ressource
ISSN:
2001-3078
,
2001-3078
DOI:
10.1080/20013078.2018.1438727
Sprache:
Englisch
Verlag:
Wiley
Publikationsdatum:
2018
ZDB Id:
2683797-3
