Milk fat miRNome changes in response to LPS challenge in Holstein cows

Cows were fed a lactation diet at ad libitum intake (n = 6). At 27±3 days in milk, cows were injected with 50 µg of LPS E. coli in one healthy rear mammary quarter. Milk samples were collected just before LPS challenge (LPS-) and 6.5 h after LPS challenge (LPS+) from the same cows. Microarray analysis was performed using customized 8x60K ruminant miRNA microarrays to compare LPS- to LPS+ miRNome. MiRNome comparison between LPS- and LPS+ identified 37 differentially abundant miRNAs (q-value ≤ 0.05) Six miltiparous cows were fed ad libitum intake of a lactation diet. At 27±3 days in milk, one healthy rear mammary quarter of all cows was injected with 50 µg of LPS. Residual milk in mammary gland was collected after AM milking, just before infusion (LPS-) and the end of PM milking, corresponding to 6.5 h after LPS injection (LPS+). One g of MF was mixed with 2.0 mL of TRIzol LS solution, then 600 µL of chloroform were added before centrifugation at 12,000 x g for 20 min at 4°C. The upper aqueous layer was precipitated using 500 µL of isopropanol overnight. The pellet obtained by centrifugation at 12,000 x g for 10 min at 4°C was washed and RNA was dissolved in 50 μL of RNase-free water.