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MiRNA marker related to Hu sheep muscle cell proliferation as well as detection primer and application of miRNA marker

A muscle cell and primer detection technology, applied in DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problem of small proportion of miRNA

Inactive Publication Date: 2020-12-15
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The proportion of miRNAs whose functions have been verified and whose target genes have been determined is still very small in the existing miRNA library, and the use of bioinformatics to theoretically predict the target genes of miRNAs inevitably has errors, which must be verified through experiments and supplemented by relevant literature data for further analysis

Method used

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  • MiRNA marker related to Hu sheep muscle cell proliferation as well as detection primer and application of miRNA marker
  • MiRNA marker related to Hu sheep muscle cell proliferation as well as detection primer and application of miRNA marker
  • MiRNA marker related to Hu sheep muscle cell proliferation as well as detection primer and application of miRNA marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Screening of miRNAs related to YAP1 and functional identification of their mimics and inhibitors

[0051] 1. Bioinformatics prediction

[0052] According to online bioinformatics software such as Findtar3, miRBase, RNA22, and miRanda, sheep miRNAs with potential target relationships in the 3'UTR region of sheep YAP1 gene were predicted, and miRNA-29a was screened as a candidate miRNA for subsequent functional verification based on the referenced literature.

[0053] 2. Preparation of RNA samples (using miRNA kit for operation)

[0054] The cells used in the experiment came from the longissimus dorsi muscle of the newborn Hu sheep in the Suzhou breeding farm, and the isolation and identification of Hu sheep muscle satellite cells have been completed in the early stage. The RNA extraction steps are as follows:

[0055] (1) Cell Recovery and Culture

[0056] Take the cryopreservation tube out of the liquid nitrogen and quickly put it into a 37°C water bath to ...

Embodiment 2

[0076] Embodiment 2 double luciferase reporter gene detection

[0077] 1. Construction of YAP1 target fragment vector

[0078] Upstream and downstream primers containing restriction endonuclease sites (the sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4) were designed, and the total length of the amplification of the primers was 535bp.

[0079] Specific steps are as follows:

[0080] (1) PCR reaction system (20uL): 2×GC 10.5uL, dNTP mix 4uL, upstream and downstream primers (the sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4) (10uM) each 1uL, LA enzyme 0.2ul, DNA template 1uL (about 100ng), make up 20uL system with sterilized water. The reaction conditions are (drop-down PCR): pre-denaturation at 95°C for 5min, denaturation at 94°C for 10s within the cycle, annealing at 1°C per cycle from 62°C, extension at 72°C for 45s, 33 cycles; extension at 72°C for 10min; amplified product Store in a 4°C refrigerator. After the PCR reaction, 5uL of the PCR prod...

Embodiment 3

[0119] Example 3 Fluorescent quantitative detection of the effect of miRNA-29a on the relative expression of YAP1 and muscle-related genes

[0120] 1. Total RNA extraction

[0121] by every 10cm 2Add 1mL lysate RZ to the area to collect the satellite cells of goat muscle transfected for 36 hours, blow it with a gun until the solution is transparent; let stand at room temperature for 5min; add 200uL chloroform, shake vigorously on the shaker for 15s and let stand at room temperature for 3min; 4 Centrifuge at 12000rpm for 10min at ℃, transfer the upper aqueous phase to a new 1.5mL enzyme-free tube; add 0.5 times the volume of absolute ethanol, mix well and transfer to RNase-Free adsorption column CR3, centrifuge at 12000rpm at 4℃ for 30s, discard Add 500uL protein-removing solution RD, centrifuge at 12000rpm at 4°C for 30s, discard the waste liquid; add 500uL rinse solution RW, let stand at room temperature for 2min, centrifuge at 12000rpm at 4°C for 30s, discard the waste liqu...

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Abstract

The invention provides a miRNA marker related to Hu sheep (a kind of sheep mainly raised in Zhejiang Province) muscle cell proliferation and a detection primer and application of the miRNA marker. ThemiRNA marker is miRNA-29a, and the sequence of the miRNA marker is SEQ ID NO. 2. The miRNA targeting YAP1 is predicted through bioinformatics, then a dual luciferase report system, RT-qPCR, CCK-8 andWestern blot technologies are utilized to further verify that the expression of the miRNA-29a plays a significant down-regulation role in the Hu sheep muscle cell proliferation process, and the provided miRNA-29a can be used as a biomarker to be applied to identification of proliferation of Hu sheep muscle cells.

Description

technical field [0001] The invention relates to a miRNA marker related to the proliferation of Hu sheep muscle cells, detection primers and applications thereof, in particular to the application of miRNA-29a in the process of Hu sheep muscle cell proliferation, and belongs to the technical field of animal husbandry and veterinary medicine. Background technique [0002] Hu sheep is a world-famous sheep breed. It not only has outstanding reproductive performance, but also has a high meat-to-bone ratio, tender and juicy meat, and is deeply loved by the public. However, due to the poor slaughter performance of Hu sheep, the development and utilization of Hu sheep meat quality traits are constantly restricted. Therefore, it is of great significance to scientifically carry out research on the regulation mechanism of meat quality traits of Hu mutton. [0003] With the rapid development of molecular biology and bioinformatics, the research on animal muscle growth and development ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/113C12N15/11A01K67/02
CPCA01K67/02C12Q1/6888C12Q2600/124C12Q2600/158C12Q2600/178
Inventor 孙伟张卫博王善禾王利宏苏锐曹修凯
Owner YANGZHOU UNIV
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