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Application of LRRC4 gene promoter zone methylation detection to glioma diagnosis and detection system

A gene promoter region and promoter region technology, applied in the development field of the methylation detection system of the LRRC4 gene promoter region, can solve the problem of interfering with heredity and cannot adapt to the screening and early diagnosis of high-risk groups of human glioma problems, achieve high sensitivity, far-reaching clinical significance, generalization, and easy operation

Inactive Publication Date: 2008-11-05
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the reference standards for the diagnosis, staging and typing of glioma are based on clinical, pathological and imaging information, and are still in the stage of empirical standards, which are far from being suitable for human glioma. The need for high-risk population screening and early diagnosis
Due to the essential characteristics of "single gene multiple targets" of genetic tumor markers and the high heterogeneity at the cell level of solid tumors, the detection data of genetic (mutation, LOH and SNP) and expressive molecular markers are seriously interfered, The clinical application of biomarkers (heredity and expression) has great limitations

Method used

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  • Application of LRRC4 gene promoter zone methylation detection to glioma diagnosis and detection system
  • Application of LRRC4 gene promoter zone methylation detection to glioma diagnosis and detection system
  • Application of LRRC4 gene promoter zone methylation detection to glioma diagnosis and detection system

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Preparation and operation of brain tissue-specific gene LRRC4 methylation detection system

[0026] The brain tissue-specific gene LRRC4 methylation detection system of the present invention can quickly and accurately determine the methylation status of CpG islands by using the PCR method. According to the working principle of the system, the system contains 2 types of kits: DNA bisulfite modification kit and LRRC4 methylation-specific PCR amplification kit.

[0027] The system works in 3 steps:

[0028] (1) DNA bisulfite modification kit preparation and operation steps

[0029] Principle: DNA samples are treated with bisulfite, which is characterized by the ability to convert cytosine in unmethylated DNA sequences into uracil, while cytosine in methylated DNA sequences is protection without transformation.

[0030] Contents: The kit mainly includes: Reagent A solution--3M NaOH 200μl, B solution--10mM hydroquinone (hydroquinone) 1ml, C solution--3.6M sodiu...

Embodiment 2

[0069] Example 2: Specificity, sensitivity and stability detection of the brain tissue-specific gene LRRC4 methylation detection system

[0070] Collect 5ml of whole blood separated plasma from 30 patients with glioma before surgery (stored in a -70°C refrigerator), and collect glioma tissue samples from the same patient (frozen with liquid nitrogen), and collect 10 normal brain tissues and paired Plasma was used as normal control.

[0071] Take 1ml of plasma to extract free gDNA in plasma according to the extraction steps of Cell-Free DNA Isolation Kit of Shanghai Huashun Biological Company. Take 30 mg of brain tumor or normal brain tissue and cut it into pieces, add 200 μl TE buffer, 400 μl cell lysate (10 mmol / L Tris-Cl (pH 8.0), 1 mmol / L EDTA (pH 8.0), 0.5% SDS), and add 20 μl of proteinase K (20 mg / ml), digested overnight at 55°C. Boil for 10 minutes, inactivate proteinase K, centrifuge at 6000rpm for 10min, take the supernatant and add an equal amount of phenol: chloro...

Embodiment 3

[0077] Example 3 Clinical Application of Brain Tissue-Specific Gene LRRC4 Methylation Detection System

[0078] Collect 5ml of whole blood from 100 patients with suspected glioma diagnosed by clinical and imaging before surgery, separate the free gDNA in the plasma according to the above method, and make pathological diagnosis on the postoperative tissues of the patients. Using the brain tissue-specific gene LRRC4 methylation detection system of the present invention to detect the methylation of the LRRC4 gene promoter region of the patient's plasma gDNA.

[0079] First, use the DNA bisulfite modification kit of the present invention to carry out bisulfite modification on the plasma gDNA sample, and refer to the aforementioned step (1) for the specific operation.

[0080] Then get 2 μl of sample DNA modified by bisulfite, and add respectively methylation and non-methylation specific primers (each 2 μl ) PCR amplification system (23 μl), put it on the PCR instrument and amplif...

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Abstract

The invention discloses an application of the LRRC4 gene promoter methylation detection to the glioma diagnosis and a detecting system thereof. The brain tissue specificity expressive gene LRRC4 is the glioma specificity methylate alienation gene, thereby indicating that the LRRC4 gene promoter methylation detection can be applied to the early period diagnosis of the glioma and the LRRC4 gene promoter methylation detecting system can be prepared by comprising a DNA bisulfite modification kit and an LRRC4 promoter methylation specificity PCR augmentation kit. The detecting system detects the specificity of the plasma LRRC4 promoter methylation state up to 92 percent, has high sensitivity, and can detect the plasma LRRC4 promoter methylation state of people quantificationally, thereby diagnosing the glioma of the patient rapidly without wound in the early time.

Description

technical field [0001] The invention relates to the application of hypermethylation of the promoter region of brain tissue-specific expression gene LRRC4 in the early diagnosis of glioma and the development of a methylation detection system for the promoter region of LRRC4 gene used for the early diagnosis of glioma. Background technique [0002] Statistics from the World Health Organization in 2007 showed that among nervous system tumors, glioma had the highest incidence rate, accounting for about 40.49%, and the peak age of onset was between 30-40 years old, or 10-20 years old, which seriously affected social productivity. For decades, although various improved treatment and diagnosis methods have been tried, including surgical treatment, chemotherapy, radiotherapy and imaging diagnostic techniques, etc., the survival time of malignant glioma has not been significantly improved. Therefore, screening and early diagnosis of high-risk groups for human glioma, so as to carry o...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 李桂源张祖萍武明花李夏雨李小玲刘恩伊李丹陈攀
Owner CENT SOUTH UNIV
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