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cRNA (complementary ribonucleic acid) in situ hybridization probe of neuropeptide Y and preparation method of cRNA in situ hybridization probe

An in situ hybridization and neuropeptide technology, applied in the field of biomedicine, can solve the problem that the detection of mRNA level is still blank, and achieve the effect of clear specificity

Inactive Publication Date: 2013-09-11
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Morphological detection of neuropeptide Y mRNA levels in animals is still blank

Method used

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  • cRNA (complementary ribonucleic acid) in situ hybridization probe of neuropeptide Y and preparation method of cRNA in situ hybridization probe
  • cRNA (complementary ribonucleic acid) in situ hybridization probe of neuropeptide Y and preparation method of cRNA in situ hybridization probe

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Embodiment Construction

[0026] 1. Design of primers and probes:

[0027] According to the mRNA sequence of neuropeptide Y provided by Gene bank, design its specific primer pair, and use this pair of primers to amplify the fragment of neuropeptide Y of 475bp as the specific probe sequence of neuropeptide Y; the primer pair sequence is as follows:

[0028] Upstream primer: 5'-tcacagaggcacccagagcag-3';

[0029] Downstream primer: 5'-aatggggcggagtccagcctag-3'.

[0030] The probe sequence is shown in SEQ ID No.1.

[0031] Two, the construction of the plasmid of the cRNA probe of neuropeptide Y:

[0032] 1. The mouse cDNA was amplified by PCR with the designed primer pair;

[0033] 2. Recover the PCR amplification product with Tiangen's glue recovery kit;

[0034] 3. Ligate the recovered product into the pGEM T-easy vector under the action of T4 ligase;

[0035] 4. Transform the ligated plasmid into competent DH5α bacteria, culture the bacteria and send them to Invitrogen for sequencing.

[0036] 3....

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Abstract

The invention relates to a cRNA (complementary ribonucleic acid) in situ hybridization probe of neuropeptide Y. The sequence of the cRNA in situ hybridization probe is as shown by SEQ ID No.1. A preparation method of the cRNA in situ hybridization probe comprises the following steps of: (1) designing a specific primer pair of the neuropeptide Y according to an mRNA (messenger ribonucleic acid) sequence of the neuropeptide Y provided by the Gene bank, and amplifying out a 475bp fragment of the neuropeptide Y by using the primer pair to serve as a specific probe sequence of the neuropeptide Y; (2) establishing plasmids for expressing the cRNA probe of the neuropeptide Y; and (3) preparing the cRNA in situ hybridization probe of the neuropeptide Y. By adopting the cRNA in situ hybridization probe of the neuropeptide Y, a model of a live animal is subjected to perfusion, material obtaining and slicing, and a digoxin marked cRNA probe is specifically combined with the mRNA of the neuropeptide Y in tissues, so that the changes of the mRNA level of the neuropeptide Y in the tissues can be conveniently and accurately observed. The cRNA in situ hybridization probe of the neuropeptide Y has definite specificity for displaying the mRNA level of the neuropeptide Y, thereby achieving the detection aim.

Description

technical field [0001] The invention relates to biomedicine, in particular to a cRNA in situ hybridization probe of neuropeptide Y and a preparation method thereof. Background technique [0002] Neuropeptide Y is one of the most abundant neuropeptides widely present in the central and peripheral systems of mammals. In the central nervous system, its functions mainly include: promoting food intake, promoting energy conversion into fat, anti-anxiety, anti-epileptic, alleviating pain, and lowering blood pressure. It is usually used as the target of dieting drugs, but in the peripheral system, it, together with glucocorticoids and catecholamines, can enhance the stress response and play an important role in stress-induced cardiovascular diseases. Neuropeptide Y exerts its function mainly by binding to its receptors. There are currently five subtypes of NPY receptors in mammals reported, four of which function in the human body, and Y1 and Y5 play roles in stimulating diet. fun...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10
Inventor 王红涛胡泽岚郑静熊志奇李奎祖勇沈娇宁刘霁纬朱志川刘永杰
Owner EAST CHINA UNIV OF SCI & TECH
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