Exosome macromolecule dosages
EVerZom provides exosome dosage services to quantify proteins, nucleic acids and lipids.
Qubit Fluorometric Quantification
Purity of Extracellular Vesicles (EVs) productions are partly defined by the ratio of EVs / µg of proteins (Webber & Clayton, Journal of Extracellular Vesicles 2013). Moreover, EV content in RNA (mRNA, miRNA,…) is of particular interest as RNA loaded in EVs is able to enter in target cells able. Qubit fluorometer is a fluorometric quantification device using specific, fluorescent dyes able to emit when bound to target molecules (DNA, RNA, or proteins). Qubit fluorometer is more accurate and sensitive than UV absorbance. The minimum concentrations detected by Qubit fluorometers are 10 pg/µL of DNA, 250 pg/µL of RNA and 12.5 µg/mL of proteins, using 1 to 20 µL of the tested solution.
Micro BCA & Bradford assays
Proteins are both a major component and a contaminant of EV preparations. Different protein assays can be used to quantify the total protein content. Bradford assay is based on complexation of aromatic amino acids with Coomassie blue, inducing a color shift followed by spectroscopy. Its limit of protein quantification is from 5 µg/mL to 120 µg/mL. To increase sensitivity, the micro BCA (bicinchoninic acid) assays consists to follow the amine-dependent reduction of Cu(II) in Cu(I). Typically, it permits to quantify proteins from 0.5 µg/mL. However, it is more sensitive to contaminants, such as free amino acids from the culture medium. Both BCA and bradford require a sample volume of 100 µL.
Nanodrop spectrophotometer is a UV-visible spectrophotometer for micro-volume (0.5 to 2 µL) permitting to quickly (few seconds) assess both the concentration and the quality of DNA, RNA or proteins in a solution. UV absorbance bands from the different biomolecules being not specific, it is limited to purified samples, after protein or nucleic acid extraction.
Nucleic acid electrophoresis (Agilent Tapestation & Bioanalyzer)
Electrophoresis is a common technique to separate nucleic acids according to their migration speed across a gel induced by an electric field. The migration depends on the electric charge of the DNA/RNA fragment which is dependent of its size. Agilent instruments are automated electrophoresis systems which provide a quality control on nucleic acid samples after their extraction, giving a very precise value of concentration for each size (miRNA, mRNA, rRNA,…)
SPV lipid assay
The lipid content is an essential part of the EVs. The best approximation of total lipid mass contained in a sample is provided by a dosage of C=C double-strand links, associated with insaturated lipid presence in EVs samples. Based on an available protocol (Visnovitz & al, Journal of Extracellular Vesicles 2019), we used the sulfo-phospho-vanillin (SPV) lipid assay with a liposome standard. The obtained value is expressed in µg lipids / ml, and can be used to estimate the EV/lipid ratio or the lipid/protein ratio, giving information on the nature of the sample.