Expression profile of miRNAs in Extracellular vesicles (EVs) obtained from Bovine granulosa cells subjected to heats stress

The study aimed to uncover the release of miRNAs via EVs and the differential release of these miRNAs from bovine granulosa cells in response to heat stress We used ovarian follicle samples obtained from local slaughterhouse. Granulosa cells were aspirated from small growing follicles (3-6 mm in diameter) and cultured for 24 hours in DMEM-F12 media + 10% Exosome-depleted FBS at 37OC. Following this, the one group of the cells were subjected to 42OC for 24 hours and the control group were left at 37OC. The spent culture medi was collected from all experimental groups. Following that, samples were centrifuged at 25000 xg for 30 minutes at 4OC to remove microvesicles. The purified cell culture medium was subjected to EVs isolation. For large scale miRNA profiling, 5ml of purified spent culture media was added with 1 ml of ExoQuick-TC (SBI System Biosciences, Inc.) and mixed very well and incubated over night at 4OC. Samples were centrifuged at 1500 xg to obtain the EVs pellet. After washing the pellet with PBS, EVs were resuspended in 500 µl PBS in -80OC until further use. Total RNA enriched with miRNAs from a total of 6 samples (three biological replicates from each treatment) were used for miRNA deep sequencing anaysis.