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Method for rapidly obtaining coding region sequence of goose ferritin heavy-chain gene and quantitatively detecting expression of gene, and primers thereof

A ferritin heavy chain and gene coding technology, which is applied in the field of rapid acquisition of goose ferritin heavy chain gene coding region sequence and quantitative detection of its expression and its primers, can solve the problem of inability to accurately detect the expression of the target gene and the inability to understand the whole process Quantitative process changes, quantitative result deviations and other problems, to achieve the effect of saving test funds, small workload and low cost

Inactive Publication Date: 2013-11-27
SICHUAN AGRI UNIV
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  • Abstract
  • Description
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Problems solved by technology

Therefore, the existing method can only obtain the copy number of the target gene fragment at the end of the PCR reaction, and cannot fully understand the changes in the quantitative process
Due to the influence of semi-quantitative electrophoresis operation, the resolution and accuracy of the gel imaging system and optical density analysis, the quantitative results will have large deviations and errors, and the expression of the target gene cannot be detected very accurately.

Method used

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  • Method for rapidly obtaining coding region sequence of goose ferritin heavy-chain gene and quantitatively detecting expression of gene, and primers thereof
  • Method for rapidly obtaining coding region sequence of goose ferritin heavy-chain gene and quantitatively detecting expression of gene, and primers thereof
  • Method for rapidly obtaining coding region sequence of goose ferritin heavy-chain gene and quantitatively detecting expression of gene, and primers thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1 Goose FTH Cloning of the full-length coding sequence of the gene.

[0021] The steps of total RNA extraction from ovarian tissue of Sichuan white goose are as follows.

[0022] (1) Take the goose ovary tissue sample out of the -80 ℃ refrigerator, grind it in liquid nitrogen, put 50-100 mg of the tissue sample into an Eppendorf tube containing 1 mL RNAiso Plus (Bao Biological Engineering Co., Ltd., Dalian) After mixing by inversion, let stand at room temperature for 10 min.

[0023] (2) Centrifuge at 12,000 g for 10 min at 4 °C, transfer the supernatant to another RNase-free Eppendorf tube, add 200 μL of chloroform, vortex and mix well, and let stand on an ice box for 15 min.

[0024] (3) Centrifuge at 12,000 g for 15 min at 4°C, absorb the upper aqueous phase, put it in another RNase-free Eppendorf tube, add an equal volume (400-500 μL) of isopropanol to the aspirated supernatant and mix well , let stand on the ice box for 10 min.

[0025] (4) Centrifuge a...

Embodiment 2

[0046] Embodiment 2 quantitative detection goose FTH Methods and primers for gene expression regulation.

[0047] The goose obtained according to embodiment 1 FTH The full-length coding sequence of the gene was designed and synthesized as the specific primer FTH-S for real-time fluorescent quantitative PCR reaction. The primer sequence is: FTH-S upstream primer: 5'CAGGGTGGTG TTCGTGGCA3'; FTH-S downstream primer: 5'CTGGATGAGC AGGTGAAGG3'.

[0048] Add 13.5 μL of sterilized deionized water, 2.0 μL of FTH-L upstream primer (10 μmol / L), and 2.0 μL of FTH-L downstream primer (10 μmol / L) into a sterile RNase-free PCR tube , 7.5 μL of template cDNA (the cDNA template obtained in Example 1), 25.0 μL of Taq DNA Enzyme Master Mix, vortexed to mix, and placed in a PCR machine after spinning. Amplify short fragments FTH (FTH-S primer) PCR reaction cycle parameters are as follows: 95 °C for 3 min; 95 °C for 10 s, 65 °C for 30 s, 72 °C for 30 s, 30 cycles, 72 °C for 10 min. After the PC...

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Abstract

The invention discloses a method for rapidly obtaining the coding region sequence of a goose ferritin heavy-chain gene and quantitatively detecting the expression of the gene, and primers thereof. The method comprises the following steps: extracting the total RNA from goose tissues, carrying out inverse transcription, and designing an FTH coding region sequence primer FTH-L (SEQ ID NO:1-2) and an FTH expression rule detection primer FTH-S (SEQ ID NO:3-4); and carrying out RT-PCR amplification, recovering and purifying the obtained product, carrying out target gene connection and conversion, and screening and sequencing a positive bacterial colony to obtain the FTH coding region sequence and the sequencing identification fluorescence quantitative PCR primers. The method has the advantages of low cost, high efficiency, obtaining of the complete coding region sequence only through one-time cloning, and rapid and accurate detection of the expression rule of the FTH in samples of different tissues and different growth periods, and lays a theoretic foundation for the clarification of the FTH structure and function, and the researches of the iron metabolism mechanism in the goose.

Description

technical field [0001] The invention relates to a method for rapidly obtaining the coding region sequence of gooseferrin heavy chain gene and quantitatively detecting its expression and primers thereof. Background technique [0002] Iron is one of the trace elements necessary for cell growth, proliferation, and maintenance of biological body functions. It plays an important role in cellular DNA synthesis, electron transfer, and oxygen transport. However, iron overload can potentially induce the generation of oxygen free radicals, leading to cellular damage. Therefore, the maintenance and regulation of cellular iron homeostasis has become the central link in the current research on iron metabolism. Ferritin is a highly conserved multifunctional, multi-subunit protein that plays a dual role in maintaining intracellular iron homeostasis: on the one hand, if the iron content in the cell increases, ferritin will sequester The free iron in the cell, the excess iron in the cell i...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C12N15/11
Inventor 康波姜冬梅白林王迅赵玲王林杰王艳何珲马容
Owner SICHUAN AGRI UNIV
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