Bovine serum microRNA molecular markers of fatty liver disease and perinatal-related metabolic diseases of dairy cows
A technology of molecular markers and fatty liver, applied in the field of molecular genetics, to achieve the effect of improving accuracy, avoiding economic loss, and simple and fast method
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Embodiment 1
[0049] Example 1. Pre-diagnosis of fatty liver disease in dairy cows during the peripartum period and selection of extreme diseased individuals and normal individuals
[0050] 1. Sample collection
[0051] The experimental animals came from farm A in Taian, Shandong and farm B in Qingdao, Shandong. A total of 131 Chinese Holstein cows were selected in the early lactation period (taking the delivery date as the first day, and the lactation days were 35.5±21.9 days). Free access to food and water. On the day of sample collection, 20ml of blood was collected before feeding in the morning. After standing at room temperature without anticoagulation for 1 hour, the serum was collected by centrifugation at 3000rpm at 4°C, and stored at -80°C after aliquoting.
[0052] 2. Reagents
[0053] Free fatty acid (NEFA) test kit (Nanjing Jiancheng Bioengineering Institute); aspartate aminotransferase IFCC method detection kit (Sichuan Mike Technology Co., Ltd.); blood glucose (GOD-PAP) met...
Embodiment 2
[0066] Example 2. Differential expression of cow serum miRNA in the control group and the test group
[0067] 1. Serum miRNA extraction
[0068] Take 400 μl of the selected dairy cow serum, add 400 μl of lysate MZ, oscillate and mix for 30 sec, place at room temperature for 5 min, add internal reference cel-miR-39-3p, and extract miRNA according to the instructions of miRcute miRNA extraction and isolation kit. Extracted miRNA was dissolved in 15 μl RNase-Free ddH 2 O. Take 1μl sample and measure OD 260 / OD 280 and concentration, OD 260 / OD 280 Should be between 1.8-2.0. Store at -80°C for later use.
[0069] 2. miRNA reverse transcription
[0070] cDNA was synthesized by reverse transcription of miRNA using TaKaRa's PrimeScript TM miRNA RT-PCR Kit, operated according to the instructions. Reverse transcription system (20μL): 2×miRNA Reaction Buffer Mix 10μl, 0.1% BSA 2μl, miRNA PrimeScript RT Enzyme Mix 2μl, miRNA (1μg) and RNase Free ddH 2 O total 6μl; reaction ...
Embodiment 3
[0078] Example 3 Accuracy detection of miR-16-5p and miR-29c-3 as molecular markers for fatty liver diagnosis
[0079] In order to test the accuracy of the diagnosis, the ROC curves command in the MedCalc 11.4.2.0 software was used for analysis, and the ROC (receiver-operating characteristic, ROC) curve was constructed to judge the deviation between the true positive rate and the false positive rate. The specific method is: in the first line of the Date window, name the variable names as diagnosis and test. In diagnosis, fatty liver is 1, non-fatty liver is 0, and test is the corresponding 2. -△△CtValue, using the Statistics / ROCcurves / ROC curve analyze process, using test as a variable and diagnosis as a classification variable for ROC curve analysis. The area under the ROC curve (AUC) was used to evaluate the sensitivity (sensitivity) and specificity (specificity) of miR-16-5p and miR-29c-3 as molecular markers for the diagnosis of fatty liver in cows, respectively. The sign...
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