MicroRNA (micro ribonucleic acid) molecular marker miR-23a for quickly detecting quality of oocytes of sows and application of microRNA molecular marker miR-23a
A mir-23a, oocyte technology, applied in DNA/RNA fragments, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of sow injury, non-instructive production, staying in the laboratory, etc.
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Embodiment 1
[0057] Example 1 Microarray method to compare the differences in miRNA expression profiles in cumulus oocytes with low developmental potential
[0058] The commercial hog ovaries were collected from the slaughterhouse, and the ovaries were transported back to the laboratory as soon as possible in sterile saline at 37 °C. Use a sterile syringe with a 20-gauge needle to extract follicles with a diameter of 2-6mm on the surface of the ovary, and slowly inject the extracted follicle fluid into a high-pressure sterilized centrifuge tube to allow the oocytes to settle naturally for 10 minutes, and then use a pipette to slowly Aspirate the follicle supernatant slowly, and transfer the precipitate after removing the supernatant to a 60mm culture dish containing PBS buffer. Divide the cumulus oocytes into two types with a mouth pipette under a stereomicroscope and transfer them to 1ml centrifuge tubes, and store them at -70°C for testing. Classification criteria: low developmental pot...
Embodiment 2
[0069] Embodiment 2 verifies the result of the chip with real-timePCR technology
[0070] The commercial hog ovaries were collected from the slaughterhouse, and the ovaries were transported back to the laboratory as soon as possible in sterile saline at 37 °C. Use a sterile syringe with a 20-gauge needle to extract follicles with a diameter of 2-6mm on the surface of the ovary, and slowly inject the extracted follicle fluid into a high-pressure sterilized centrifuge tube to allow the oocytes to settle naturally for 10 minutes, and then use a pipette to slowly Aspirate the follicle supernatant slowly, and transfer the precipitate after removing the supernatant to a 60mm culture dish containing PBS buffer. Divide the cumulus oocytes into two types with a mouth pipette under a stereomicroscope and transfer them to 1ml centrifuge tubes, and store them at -70°C for testing. Classification criteria: low developmental potential cumulus oocytes: oocytes with uniform cytoplasm, only a...
Embodiment 3
[0092] Example 3 Using real-timePCR technology to detect the expression level of miR-23a in the serum of sows with poor oocyte quality
[0093] Serum sample collection: Collect 10ml of blood on an empty stomach the next morning when sows appear standing reflex, centrifuge at 2000-3000rpm for 18-20min, draw the supernatant into RNase-free centrifuge tubes, aliquot 400μl, and freeze at -80℃ until Measurement.
[0094] Research sample selection criteria: Landrace sows with less than 5 litters in two consecutive parities, body condition score (2.5-3.5), and normal and healthy food intake are defined as the poor oocyte quality group; Healthy Landrace sows with the same parity, two consecutive parities with 10-13 litters, body condition score (2.5-3.5), and normal feed intake were defined as the normal control group. A total of 100 samples were collected, 50 of which were from the low litter size group and 50 from the normal control group.
[0095] Extraction of serum microRNA
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