This protocol allows us to use a reduced number of ticks which reduces the cost of colony maintenance and decreases the number of vertebrae animals needed for tick feeding. This protocol requires approximately 20 ticks. However, the main advantage aside from using a small sample size is that this protocol can be applied to multiple tick species and different life stages.
Aside from tick dissections or the application towards host-parasite interactions, the effect of pathogen infection on microRNA secretion and the effect of physiological changes on microRNA secreted by ticks. To begin, prepare vesicle-free media by adding fetal bovine serum, tryptose phosphate broth, 10%lipoprotein cholesterol concentrate, 5%sodium bicarbonate, HEPES, and L15C300 medium into a 26.3 milliliter polycarbonate centrifuge bottle and adjust the volume as required. Now ultracentrifuge the medium at 100, 000 times G at four degrees Celsius for 18 hours.
Carefully remove the supernatant without disturbing the pellet and transfer the supernatant to a fresh centrifuge tube. Ultracentrifuge the tube once again. Take the supernatant and pass it through a 0.22 micrometer filter to remove contaminants.
Pipette the supernatant into a 50 milliliter centrifuge tube and store it at minus 20 degrees Celsius until required or up to three years. Add an appropriate amount of antibiotics to 500 microliters of the vesicle-free medium in each well of a 24-well culture plate. Now, add PBS with rifampicin to the wells that do not contain vesicle-free medium to prevent bacterial growth.
Place the ticks on a glass slide with double-sided carpet tape under a dissecting microscope. Before dissection, add PBS containing rifampicin to the tick and using four millimeter Vannas scissors, make an incision of approximately one millimeter on the side of each female. Now, remove the tick's dorsal side and salivary glands, then put 20 to 40 tick salivary glands in 500 microliters of vesicle-free medium added to a single well in a 24-well tissue culture treated plate.
Incubate the salivary gland samples at 32 degrees Celsius for 24 hours to allow the secretion of the extracellular vesicles. At the end of the incubation, pipette all the medium containing the salivary glands into a 1.5 milliliter microcentrifuge tube and centrifuge at 300 times G at four degrees Celsius for 10 minutes. Transfer the supernatant into a new 1.5 milliliter microcentrifuge tube.
Resuspend the pellet containing the salivary glands in RNA later and store at minus 80 degrees Celsius until used or indefinitely. Now, remove the cellular debris by centrifuging the supernatant at 2, 000 times G at four degrees Celsius for 10 minutes and pipette the supernatant into a new 1.5 milliliter microcentrifuge tube. Next, centrifuge at 10, 000 times G for 30 minutes at four degrees Celsius to remove apoptotic bodies and larger extracellular vesicles and transfer the supernatant to a fresh 1.5 milliliter microcentrifuge tube.
Then assemble a one micrometer nylon syringe filter on a 10 milliliter syringe and keep it on an ultracentrifuge tube. Then add the sample and fill the syringe with 10 milliliters of PBS. Pass the supernatant through the filter into the tube.
After filtering and balancing the tubes, place them into a 70 Ti rotor and ultracentrifuge the tubes at 100, 000 times G for 18 hours at four degrees Celsius. The concentrated extracellular vesicles form a pellet after ultracentrifugation. Remove the supernatant without disturbing the pellet and resuspend the pellet in PBS.
Pipette 500 microliters of the extracellular vesicle into a 300 K centrifugal filter and centrifuge at 8, 000 times G for 10 minutes at ambient temperature and repeat this procedure until all extracellular vesicles are filtered. Finally, add 400 microliters of sterilized PBS to the column and mix thoroughly to remove extracellular vesicles attached to the membrane. Then put the sample into a 1.5 milliliter DNAase RNAse free tube.
The concentration and size of extracellular vesicles varied depending on the gender and the life stage of the ticks in between the biological replicates for females, males, and nymphs. For all combined biological replicates, similar results were observed. Analysis of small RNAs isolated from the salivary gland showed bands corresponding to ribosomal RNA and microRNAs.
However, the ribosomal RNAs were absent in the vesicles isolated from the salivary glands, and microRNA samples purified from extracellular vesicles showed more degradation. After enrichment, the microRNA samples purified from EVs showed minimal degradation and sufficient microRNA concentration for cDNA library synthesis. The expected band size of approximately 150 base pairs after cDNA library preparation was obtained signifies small RNA cDNA libraries.
During vesicle-free preparation, ensure to follow the steps to prevent external vesicle contamination. During well plate preparation, make sure to add antibiotics to prevent bacterial contamination from the tick microbiome. The microRNAs extracted using this protocol can be used for profiling and functional experiments to validate microRNA roles such as predicting microRNA pathway targets, inhibition, and mimicry experiments.
