Method for screening and identifying spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease (MJD) molecular marker MicroRNAs (miRNAs) capable of regulating and controlling expression of ATXN3 gene

A molecular marker and gene expression technology, applied in the field of screening and identification of SCA3/MJD molecular marker miRNAs that can regulate the expression of ATXN3 gene

Inactive Publication Date: 2013-10-09
XIANGYA HOSPITAL CENT SOUTH UNIV
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Problems solved by technology

[0007] However, adjustable ATXN3 Construction of screening and identification methods for miRNAs in gene expression, and determination of specific involvement in regulation ATXN3 The miRNAs and their targets of gene expression have not been reported at home and abroad, and further research is needed

Method used

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  • Method for screening and identifying spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease (MJD) molecular marker MicroRNAs (miRNAs) capable of regulating and controlling expression of ATXN3 gene
  • Method for screening and identifying spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease (MJD) molecular marker MicroRNAs (miRNAs) capable of regulating and controlling expression of ATXN3 gene
  • Method for screening and identifying spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease (MJD) molecular marker MicroRNAs (miRNAs) capable of regulating and controlling expression of ATXN3 gene

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Embodiment Construction

[0062] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0063] refer to Figure 1~Figure 8 :

[0064] (1) Prepare 5-10ml of anticoagulant blood containing SCA3 / MJD and anticoagulant blood without SCA3 / MJD, separate the serum and store it at -80°C for later use;

[0065] (2) TRIzol method extraction step (1) Total RNAs containing miRNA in serum:

[0066] Take out the serum from the -80°C refrigerator, and after thawing, take 0.25ml of each serum sample and 2~8ul polypropylene carrier, mix it with 0.75ml TRI Reagent BD, close the cap tightly, shake it by hand, and mix the mixture Incubate at room temperature for 5 minutes; add 0.2ml of chloroform to the mixture, cap the tube tightly, and shake vigorously for 15 seconds. Let stand at room temperature for 5 minutes, centrifuge at 12,000 g at 4°C for 15 minutes, and the mixture is divided into three layers, the lower layer is a red phenol-chloroform phase,...

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Abstract

The invention discloses a method for screening and identifying spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease (MJD) molecular marker MicroRNAs (miRNAs) capable of regulating and controlling the expression of an ATXN3 gene. The method comprises the following steps: obtaining differential expression profile of miRNAs by using miRNA microarray chip technique; further screening and verifying by using a bioinformatics method and a qRT-PCR technique to obtain the miRNAs expressed by regulated ATXN3 gene; determining the target action relation and mechanism of action of the miRNAs and ATXN3 by using qRT-PCR technique and Westernblot; determining target action relation and specific binding site of the miR-25 and ATXN3 gene by using luciferase reporter gene detection technique so as to provide basis for researching and developing diagnostic kit related to SCA3/MJD disease.

Description

technical field [0001] The present invention relates to a controllable ATXN3 Screening and identification methods of SCA3 / MJD molecular marker miRNAs expressed by genes. Background technique [0002] Spinocerebellar ataxia (Spinocerebellar ataxia, SCA) is one of the main neurodegenerative diseases in humans, among which spinocerebellar ataxia type 3 (Spinocerebellar ataxia type 3 / Machado-Joseph disease, SCA3 / MJD) is the most common. disease-causing gene ATXN3 (MJD1) The encoded protein ataxin-3 is a cytoplasmic protein containing polyglutamine (polyQ). Ataxin-3 proteins containing abnormally extended polyQ peptide chains can selectively accumulate in specific areas of the nervous system (cerebellum, brainstem, spinal cord, etc.) to form neuronal intranuclear inclusions (NIIs) and cause neuronal death. The current research has confirmed that the cytotoxicity of abnormally expanded polyQ mutant proteins triggers the occurrence and development of such diseases. Although th...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 江泓黄凤珍张莉师玉亭唐北沙
Owner XIANGYA HOSPITAL CENT SOUTH UNIV
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