Isolation and Characterisation of EV (and Cell) Molecular Contents

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Isolation of RNA

To extract RNA from cells, an aforementioned cell pellet from an aliquot of a 1 litre YPD culture was thawed on ice and 500 μl of TRIzol (Invitrogen, Thermo Fisher Scientific) was added. Zirconia beads (RiboPure-Bacteria kit, Life Technologies) were added to a 2 ml sterile RNAse-free Tube & Standard Cap Assembly (SSIbio), up to the 0.5 ml line, and the cell- TRIzol mixture was vortexed and added to this tube. Cell lysis was ensured using the Omni Bead Ruptor 24 at 3.5 m/s for two 2-minute cycles, with a 1-minute incubation on ice between the cycles. The resulting bead-lysate mixture was transferred to a 1.5 ml RNAse-free Lobind microcentrifuge tube (Eppendorf) and spun at 10,000x g for 5 minutes at 4°C. The supernatant was then transferred to another 1.5 ml RNAse-free Lobind microcentrifuge tube and 50 μl of homogenate additive (from a miRvana™ miRNA Isolation Kit (Thermo Fisher Scientific)) was added.
RNA was then isolated from the disrupted cells, as well as 200 μl of 2 l crude EV preparations or qEV fractions added to 600 μl of TRIzol® LS (Thermo Fisher Scientific) in 1.5 ml RNAsefree Lobind microcentrifuge tubes. 100 μl of chloroform (Sigma-Aldrich) and 2 μl of glycogen (20 μg/μl, Invitrogen, Thermo Fisher Scientific) were added to all samples, and the tubes were shaken and incubated for 10 minutes on ice before being spun at 10,000x g for 10 minutes at 4°C to layer the mixture. The upper aqueous phase was collected and mixed with 1.25x volume of 100% ethanol. RNA was further purified from this solution using a miRvana™ miRNA Isolation Kit, according to the manufacturer’s protocol for isolating total RNA. Briefly, the mixture of aqueous phase and ethanol was eluted through a miRvana™ column filter in a collection tube by centrifugation at 10,000x g for 15 seconds. The filtrate was discarded and 700 μl of Wash Solution 1 was added to the column, before repeating the 15-second centrifugation and removal of filtrate. This was followed by twice adding 500 μl of Wash Solution 2/3, centrifuging at 10,000x g for 15 seconds both times and discarding the filtrate.
The column was then centrifuged at 10,000x g for 1 minute. The flow-through was discarded and RNA in the column was eluted by addition of 35 μl of 95°C diethyl pyrocarbonate (DEPC)- treated water (Thermo Fisher Scientific), followed by a 15-second centrifugation at 10,000x g. The resulting 35 μl of filtrate was then re-added to the column and centrifuged for 15 seconds at 10,000x g again to ensure elution of all RNA in the column, before being stored at -20°C for RNA quantification and profiling. All centrifugations in this procedure were carried out at 4°C.

Isolation of DNA

DNA was extracted from 2 l crude EV preparations, qEV fractions and the negative control, in addition to DPBS controls, using a QIAmp® DNA Mini Kit (QIAGEN) as per the manufacturer’s instructions. Briefly, 20 μl of sample or DPBS was added to 20 μl of proteinase K and 200 μl of buffer AL. This solution was incubated at 56°C for 10 minutes, briefly centrifuged at 6000x g and added to 200 μl of 100% ethanol. The resulting solution was again briefly centrifuged, before being eluted through a QIAmp spin column in a collection tube by centrifugation at 6000x g for 1 minute. The filtrate was discarded and 500 μl of buffer AW1 was added to the spin column, before repeating the 1-minute centrifugation and removal of filtrate. 500 μl of buffer AW2 was then added, and the column was centrifuged at 15,000x g for 3 minutes, and then again at 6000x g for 1 minute after discarding the filtrate. The flowthrough was discarded and DNA in the column was eluted by addition of 50 μl of buffer AE, followed by a 5-minute incubation at room temperature and a 1-minute centrifugation at 6000x g. 50 μl of filtrate containing the eluted DNA was stored at -20°C for quantification and profiling. All centrifugations in this procedure were carried out at room temperature.

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Quantification of isolated RNA and DNA

RNA extractions from 2 l crude EV preparations, qEV fractions, cells and the negative control, and DNA extractions from crude EV preparations, qEV fractions, the negative control and the DPBS controls, were subjected to fluorometric quantitation of their respective nucleic acid using RNA or dsDNA HS (High Sensitivity) Assay kits (Thermo Fisher Scientific), respectively, according to the manufacturer’s instructions, with Qubit® assay tubes (Life Technologies) and a Qubit® 2.0 Fluorometer (Invitrogen, Thermo Fisher Scientific). A maximum of 20 μl of sample was used. The RNA extractions from cells were diluted 1 in 100 with UPW prior to this analysis.

Chapter 1. Introduction
1.1. Importance of Pathogenic Fungi
1.2. Candidiasis
1.3. Candida albicans
1.4. Candida auris
1.5. Microbial Extracellular Vesicles
1.6. Extracellular Vesicles from Fungi
1.7. Extracellular Vesicles from Candida albicans
1.8. Isolation and Purification of Extracellular Vesicles
1.9. Project Hypotheses and Aims .
Chapter 2. Materials and Methods
2.1. Fungal Stocks and Growth Conditions
2.2. Growth Curves .
2.3. Crude Extracellular Vesicle Preparations
2.4. Size-Exclusion Chromatography .
2.5. Transmission Electron Microscopy
2.6. Determining EV Sizes and Concentration
2.7. Isolation and Characterisation of EV (and Cell) Molecular Contents
Chapter 3. Results
3.1. Contamination Checkpoints
3.2. Growth Curves
3.3. High Resolution Imaging of EVs and Cells .
3.4. Quantitative Characterisation of Crude EV Preparations
3.5. Quantitative Analyses of qEV Fractions
3.6. SDS-PAGE
3.7. Protein Identification
Chapter 4. Discussion
4.1. Introduction
4.2. Transmission Electron Micrographic Evidence of EVs .
4.3. Characterisation of EVs and EV contents
4.4. Dominant Proteins in qEV Fractions .
4.5. Limitations and Future Directions .
4.6. Conclusion