LRRC55 gene methylation quantitative detection method

A technology of methylation quantification and detection method, which is applied in the field of LRRC55 gene methylation detection, can solve the problems of large sample size, complexity, and limited use of MSP, and achieve simple operation, high sensitivity, and strong accuracy Effect

Inactive Publication Date: 2011-02-09
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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Problems solved by technology

This method has the following disadvantages: (1) Since the CG is not limited to the CCGG sequence, the CG not in the sequence will be ignored; (2) only when the methylation status of the key sites related to transcription is detected, the The results of the detection method are meaningful; (3) Relatively speaking, the Southern method is more complicated and requires a large amount of sample; (4) There is a problem of false positives caused by incomplete digestion of the enzyme; (5) It is not suitable for mixing sample
The key to reliable MSP lies in the primers. The primer design requires two known regions containing multiple fully methylated or unmethylate

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  • LRRC55 gene methylation quantitative detection method
  • LRRC55 gene methylation quantitative detection method
  • LRRC55 gene methylation quantitative detection method

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Embodiment 1

[0022] Example 1: Quantitative detection of methylation degree of LRRC55 gene (GeneID: 219527) in clinical samples of patients

[0023] Sources of main reagents and instruments: QIAGEN EpiTect Bisufite KitTM kit, pMD18-T Vector, EX-taq enzyme purchased from Treasure Bioengineering (Dalian) Co., Ltd., ABI TaqmanGene Expression Master Mix purchased from Applied Biosystems, ABI 7500RealTime PCR instrument Purchased from Applied Biosystems, USA, primers were synthesized by Shanghai Bioengineering Technology Service Co., Ltd.

[0024] Steps:

[0025] 1. Preparation of standard products using gene amplification and bioengineering methods

[0026] 1. Genomic DNA Extraction

[0027] DNA was extracted by conventional phenol / chloroform method.

[0028] (1) Put 50mg of pancreatic tissue into a 5ml centrifuge tube, add 700ul of lysis buffer to make a homogenate.

[0029] (2) Add proteinase K to a final concentration of 100ug / ml, and keep at 55°C overnight until the solution becomes cl...

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Abstract

The invention belongs to the technical field of biomedicine. The abnormal methylation of genes participates in the occurrence and the development of tumors. The invention aims at overcoming the defects of the methylation sensitivity restriction enzyme method, the bisulfite sequencing method and the methylation specificity PCR (Polymerase Chain Reaction) method and providing an LRRC55 gene methylation quantitative detection method with easy and simple operation and high sensitivity. The method comprises the following steps of: taking a pancreatic cancer tissue, extracting DNA, preparing a standard curve sample of ACTB (Actin, Beta), and preparing a standard curve sample of LRRC55 by using a complete methylation template; extracting the DNA of a tissue to be tested, performing chemical modification, designing a methylation primer and probe aiming at the CpG island of a promoter region of the human LRRC55 gene, designing a BSP primer and probe aiming at the CPG island of a promoter region of human reference gene ACTB, performing real-time quantitative PCR on the DNA treated by sulfite by using the Tagman-MGB real-time quantitative PCR method, and calculating the quantitative value of the methylation degree of the LRRC55 gene. The method is rapid and accurate and is high in sensitivity.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for detecting nucleic acid, especially a method for detecting methylation of LRRC55 gene. Background technique [0002] The occurrence and development of malignant tumors is a process in which multiple genes interact and influence each other. Abnormal methylation of CpG islands is an important mechanism for the occurrence and development of this gradual process, which includes two types: hypermethylation and hypomethylation. Our previous research found that LRRC55 (leucine rich repeat containing 55, GeneID: 219527) is highly methylated in pancreatic cancer, and the methylation rate of this gene in cancer tissues is significantly higher than that in adjacent tissues. In view of the high methylation of LRRC55 in pancreatic cancer The close relationship between them prompted us to establish a new quantitative detection method for the degree of LRRC55 methyla...

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 李兆申高军吕顺莉杜奕奇龚燕芳黄浩杰王小玮
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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