Method for simultaneously detecting 17 genes

A gene and reverse transcription primer technology, applied in the field of medical molecular biology, can solve the problem of difficult to distinguish the nature and composition of plaques

Inactive Publication Date: 2011-11-23
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In particular, imaging signs are commonly used clinically to evaluate the degree of coronary artery lumen stenosis and th

Method used

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  • Method for simultaneously detecting 17 genes
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  • Method for simultaneously detecting 17 genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 The method of the present invention was used to detect the expression levels of 17 genes in 24 samples at the same time. (15 genes are cardiovascular disease-related genes, β-ACTIN and human glycerol kinase genes are reference genes for analysis)

[0057] (1) Design reverse transcription primers and PCR primers for amplifying the target gene, see Sequence 1 to Sequence 18 in Table 1 for details.

[0058] (2) Take out the prepared multiple reaction RT primer working solution and 200nmoL / L PCR primer from the refrigerator, and amplify the target fragment. The reverse transcription reaction system for 24 people is shown in Table 10.

[0059] Reverse transcription reaction system for 1024 people

[0060]

[0061] The cycle conditions are: 48°C for 1min, 42°C for 60min at, 95°C for 5min, and finally keep warm at 4°C.

[0062] (3) See Table 11 for the preparation of the PCR reaction system.

[0063] Table 1124 PCR reaction system for 24 people

[0064]

...

Embodiment 2

[0072] Example 2 The method of the present invention was used to simultaneously detect the expression levels of 17 genes in 33 samples. (15 genes are cardiovascular disease-related genes, β-ACTIN and human glycerol kinase genes are reference genes for analysis)

[0073] (1) Designing reverse transcription primers and PCR primers for amplifying the target gene, see Sequence 1 to Sequence 18 for details.

[0074] (2) Take out the prepared multiple reaction RT primer working solution and 200nmoL / L PCR primer from the refrigerator, and amplify the target fragment. The reverse transcription reaction system for 33 people is shown in Table 14.

[0075] Reverse transcription reaction system for 1433 people

[0076]

[0077] The cycle conditions are: 48°C for 1min, 42°C for 60min at, 95°C for 5min, and finally keep warm at 4°C.

[0078] (3) See Table 15 for the preparation of the PCR reaction system.

[0079] Table 1524 PCR reaction system for 24 people

[0080]

[0081] (4)...

Embodiment 3

[0089] Example 3 The method of the present invention was used to detect the expression levels of 17 genes in 48 samples at the same time. (15 genes are cardiovascular disease-related genes, β-ACTIN and human glycerol kinase genes are reference genes for analysis)

[0090] (1) Designing reverse transcription primers and PCR primers for amplifying the target gene, see Sequence 1 to Sequence 18 for details.

[0091] (2) Take out the prepared multiple reaction RT primer working solution and 200nmoL / L PCR primer from the refrigerator, and amplify the target fragment. The reverse transcription reaction system for 48 people is shown in Table 18.

[0092] Reverse transcription reaction system for 1848 people

[0093]

[0094] The cycle conditions are: 48°C for 1min, 42°C for 60min at, 95°C for 5min, and finally keep warm at 4°C.

[0095] (3) Preparation of PCR reaction system The reaction system is shown in Table 19.

[0096] Table 1948 person PCR reaction system

[0097]

...

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Abstract

The invention relates to a method for simultaneously detecting 17 genes. Sequences of mRNA of interleukin-1beta (IL-1beta, NM-000576, etc.) and sapiens glycerol kinase (NM-203391) are referred to on PUBMED; a reverse transcription primer and a PCR primer are designed; reverse transcription, PCR amplification and capillary electrophoresis are performed; and quantitative analysis is performed by a Gexp analysis system. The detection method not only comprises a quality control gene of a kanamycin resistance gene (Kan, R) for monitoring the reaction system, but also designs two amplified products for L-selectin which are used to detect the stability of the reaction system. The invention has the advantages of small sample size required, high sensitivity and accuracy. The method analyzes the properties and components of an atherosclerotic plaque by detecting the expression situation of peripheral blood related genes, and establishes a technical platform for batch screening of differential genes between cardiovascular diseases and controls.

Description

technical field [0001] The invention relates to a method for simultaneously detecting 17 genes. It belongs to the field of medical molecular biology. Background technique [0002] In recent years, the prevalence of coronary atherosclerotic heart disease (CAD) has been increasing year by year, and a considerable proportion of patients have no clinical symptoms in advance. Coronary atherosclerosis is paid clinical attention only when it develops to a certain extent and causes myocardial ischemia. The early diagnosis of coronary atherosclerosis, that is, the preclinical diagnosis that has not caused myocardial ischemia, is important for the prevention of clinical events of coronary heart disease. Whether it is early preventive intervention and treatment in high-risk groups is of great significance. Little is known about coronary atherosclerosis in asymptomatic individuals with high-risk factors. [0003] Studies have shown that metabolic disorders of blood lipids and changes...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 田亚平贾兴旺
Owner GENERAL HOSPITAL OF PLA
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