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Application of chi-miR-450-5p as miRNA marker for maturation of goat follicles

A follicle maturation and marker technology, applied in the field of molecular biology, can solve problems such as less research

Active Publication Date: 2020-05-08
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the maturity of high-throughput sequencing technology, studies on miRNA regulation of human, mouse, pig and bovine follicle development have been gradually reported in recent years, while there are few studies on miRNA regulation of goat follicle development

Method used

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  • Application of chi-miR-450-5p as miRNA marker for maturation of goat follicles
  • Application of chi-miR-450-5p as miRNA marker for maturation of goat follicles
  • Application of chi-miR-450-5p as miRNA marker for maturation of goat follicles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Sequencing identification and screening of miRNAs associated with goat follicle development

[0033] S1: Select five Leizhou goats with similar body condition and good condition (from a sheep farm in South China), and each goat is given intramuscular injection with 0.1 mg of cloprostinol sodium. After 2 days, all the black goats are in estrus; after 18 days, the second natural goat During estrus, use vaginal examination and test ram (vasectomy) for estrus identification (Jin Zhongqing, Wang Xijun, Lin Chunyan. Dairy goat estrus identification technology[J]. China Livestock and Poultry Breeding Industry, 2010,6(07):56. ). All black goats were slaughtered within 24-36 hours after estrus.

[0034] S2: After the goat is depilated and opened, immediately cut the ovary with scissors, trim the connective tissue at the edge of the ovary with scissors, wash it with 75% (v / v) ethanol once, wash it with normal saline three times, use small scissors and No. 5 tweezers F...

Embodiment 2

[0048] Example 2: Expression of chi-miR-450-5p in follicles of Chuanzhong black goat

[0049] S1: The ovary of central Sichuan black goat was collected from a slaughterhouse in Yangxi County, Guangdong Province, and a single follicle was isolated. According to the diameter of the goat follicle, it was divided into dominant follicle (>8mm and surrounded by abundant blood vessels) and small follicle (<3mm).

[0050] S2: Promega using Beijing Promega Super total RNA extraction kit is used to extract RNA from follicle samples, the steps are as follows:

[0051] 1) Put a single follicle into a nuclease-free centrifuge tube, add 500 μL RNA lysate, place on ice, and break the cells with a tissue homogenizer.

[0052] 2) Add an equal amount of RNA diluent and mix well, incubate at 70°C for 3 minutes, then centrifuge at 14000g for 5 minutes, and aspirate the supernatant.

[0053] 3) Add 0.5 times the supernatant volume of absolute ethanol, mix well, transfer to a collection column,...

Embodiment 3

[0061] Example 3: Expression of chi-miR-450-5p in goat dominant follicles and small follicle granulosa cells

[0062] S1: Ovary sample collection. The ovaries of central Sichuan black goat were collected from a slaughterhouse in Yangxi County, Guangdong Province. The connective tissue on the edge of the ovary was trimmed with scissors, washed once with 75% (v / v) ethanol, washed three times with normal saline, and placed in double antibody ( PS, 100IU / mL penicillin + 50mg / mL streptomycin) in normal saline, stored on ice and transported back to the laboratory.

[0063] S2: Cut the ovary into two halves with scissors under a stereomicroscope, avoiding the follicles, and put them in a petri dish containing DMEMF / 12 medium containing double antibodies. Use surgical scissors and No. 5 tweezers to separate follicles, and divide goat follicles into dominant follicles (>8mm and surrounded by abundant blood vessels) and small follicles (<3mm) according to the diameter of goat follicles...

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Abstract

The invention discloses an application of chi-miR-450-5p as an miRNA marker for maturation of goat follicles. The chi-miR-450-5p provided by the invention can reflect the maturation of goat follicles,and has reliability, universality and repeatability as a follicle development marker. The invention also discloses a test kit for detecting the development condition of the goat follicles. Accordingto the invention, through separation of follicle granulosa cells, the expression mode of the chi-miR-450-5p in dominant follicles and small follicle granulosa cells is verified, and a basis is provided for perfecting a goat follicle growth and development, ovulation gene regulation and non-coding regulation network.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to the application of chi-miR-450-5p as a goat follicle maturation miRNA marker. Background technique [0002] The litter size of goats is an important standard to measure their reproductive performance, and the litter size is directly related to the follicular development of goats. Follicular development is a complex biological process that is tightly regulated by multiple regulatory pathways and endocrine hormones. After puberty in animals, under the regulation of reproductive hormones, some primordial follicles on the ovary are recruited and start to develop. However, only a small number of follicles can develop into mature follicles and ovulate in the end, and most of the follicles undergo degenerative atresia during development. There are many studies on the mechanism of follicle development, and many important research results have been obtained, but there are still few stud...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/113
CPCC12Q1/6888C12Q2600/178C12Q2600/124
Inventor 刘德武赵智锋邹娴柳广斌李耀坤
Owner SOUTH CHINA AGRI UNIV
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