FastEV & Protein

FastEV gives a wide range of protein yields with good reproducibility

FastEV isolation and control EV isolation protocols were applied to plasma samples and outputs measured with Bio-Rad DC protein assay.

Methods: Total protein concentration 

FastEV isolation and control methods (ultracentrifugation, polyethylene glycol precipitation and 96-well size exclusion chromatography SmartSEC (System BioSciences) were applied for fasting and non-fasting EDTA plasma samples of 100-800 µl. Total protein was measured with DC protein assay (Bio-Rad) from isolates or original plasma using 2-4 replicates per sample. Data from different experiments were normalized per ml of original plasma used.

Figure: Total protein yields from FastEV conditions span a wide range between the lowest (UC) and highest (PEG) EV control. Protein yield results were normalized per ml of original plasma. N=10-17 experiments of FastEV, 1-5 of controls, error bars show SEM. PEG, polyethylene glycol, UC, ultracentrifugation.

Repeatable performance of FastEV conditions across donors

FastEV and control EV isolation protocols were applied to plasma samples from 3 donors and the isolates were analysed for total protein with Bio-Rad DC protein assay. Minimal inter-donor and intra-condition variation of protein yields was observed from the different FastEV conditions.

Figure: Minimal inter-donor variability of protein yields from FastEV conditions. ​A representative experiment with two technical replicates per sample, error bars show SEM. D, donor, PEG, polyethylene glycol, UC, ultracentrifugation.

FastEV & RNA

FastEV produces a range of RNA yields

FastEV isolation and control EV isolation protocols were applied to plasma samples. After total RNA extraction, the outputs were subjected to concentration and RNA profile analysis using Agilent RNA Pico kit.

Methods: RNA isolation and measurement

FastEV isolation and control EV isolation methods (ultracentrifugation, polyethylene glycol precipitation) were applied to fasting and non-fasting EDTA plasma samples of 800 µl. TotalRNA was extracted from the isolates or 200 µl of original/raw plasma with miRNEasy serum/plasma or micro kits (Qiagen) according to manufacturer’s instructions. RNA profiles and concentrations were analyzed with Agilent RNA 6000 Pico kit (Agilent Technologies). Data from different experiments were normalized per ml of original plasma used.

Figure: Total RNA yields from FastEV conditions span a wide range between the lowest (UC) and highest EV control (PEG). RNA yield results were normalized per ml of original plasma. Error bars show SEM of 3-7 independent experiments for FastEV conditions and 2-4 experiments of controls. PEG, polyethylene glycol, UC, ultracentrifugation.

RNA from FastEV is typical for liquid biopsies

RNA from all FastEV samples was similar to that of cell-free plasma and EV controls: they contained small RNA and minor quantities of ribosomal RNA. Fasting and non-fasting plasma gave similar profiles.

Figure: Total RNA from FastEV conditions consists mostly of small RNA. Representative Bioanalyzer Pico assay profiles show small RNA up to ~500 nucleotides (nt), and in most cases small peaks of ribosomal RNA. PEG, polyethylene glycol, UC, ultracentrifugation.

FastEV & EV

Unique EV recovery performance of FastEV conditions

Different FastEV conditions isolated EVs with a range of individual efficiencies ranging from >70% to <10%. The EV isolation efficiency of FastEV conditions was measured with the help of fluorescent spike-in EVs obtained from cell cultures. Fluorescent EVs were spiked into plasma prior to FastEV isolation. EV recovery was then measured in the isolates by Apogee nano flow cytometer.

Methods: measurement of EV isolation efficiency with Nano flow cytometry​

FastEV isolations were applied to EDTA plasma samples with 160 µl/well/replicate using 96-well plates. To measure EV isolation efficiency with nano flow cytometer (Apogee A50), GFP-positive EVs were spiked into plasma prior to FastEV isolations. We used CD63-GFP-positive EVs from Human Embryonic Kidney cells, courtesy of Dr. Berndt Giebel (Ludwig et al. 2018), as spike-in. Spike-in Evs were purified from cell conditioned media by ultracentrifugation. The number of purified spike-in EVs was determined by nanoparticle tracking analysis. The 100% in-put amount of spiked-in fluorescent EVs (per well) was measured by nano flowcytometer to estimate the maximum recoverable amount of EVs. The recovery (%) was calculated by dividing the fluorescent signal from the FastEV isolates with the fluorescent signal of the 100% in-put EV spike-in.

Figure: EV isolation efficiency by FastEV ranges from 10-70%. FastEV was performed and EV recoveries assessed with nano flow cytometry using 96-well plates throughout the process. The recovery (%) was calculated by dividing the fluorescent signal of the spike-in EVs in the FastEV isolates with the signal of 100% spike-in (in-put). The plot shows average recoveries (%) +/- SEM from 3-4 experiments and 2-6 technical replicates of each condition per experiment. Green fluorescent protein positive (GFP+). Schematic created with BioRender.com.