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Reagent kit for detecting epidermal growth factor acceptor third type mutant by real-time fluorescence quantitative PCR

An epidermal growth factor, real-time fluorescence quantitative technology, applied in fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., can solve problems such as disputes over the expression level of EGFRvIII, and achieve credible results and quantitative accuracy. High, improve work efficiency

Inactive Publication Date: 2008-02-27
ZHEJIANG UNIV
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  • Claims
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Problems solved by technology

[0003] So far, studies have used RT-PCR semi-quantitative analysis, Northern blotting and other detection methods to quantitatively detect EGFRvIII mRNA, but the expression levels of EGFRvIII in tumor tissues reported by different detection methods are controversial.

Method used

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  • Reagent kit for detecting epidermal growth factor acceptor third type mutant by real-time fluorescence quantitative PCR
  • Reagent kit for detecting epidermal growth factor acceptor third type mutant by real-time fluorescence quantitative PCR

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Experimental program
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Effect test

Embodiment 1

[0037] Expression of EGFRvIII detected by fluorescence quantitative PCR

[0038] 1. Materials

[0039] Trirol reagent was purchased from Invitrogen, USA, and restriction endonuclease EcoR I was purchased from NewEngland, USA.

[0040] BioLabs, pGEM-T-Easy Cloning System, M-MLV Reverse Transcriptase, Oligo(dT) 15 It was purchased from Promega Company in the United States, and Tag DNA polymerase was a product of Bioflux Company in South Korea. The Model 377 sequencer was a product of Applied Biosystems in the United States, and the real-time fluorescent quantitative PCR detector and detection system version 2.0 were purchased from Hangzhou Bioer Technology Co., Ltd.

[0041] 2. Primer design and synthesis

[0042] Taking the EGFR genome sequence (Genbank accession number: AY588246) as a reference, EGFRvIII is the splice variant form of the deletion of the 2nd to 7th exons. Primers were designed to amplify the 1st exon (Exon1, E1) and the 8th to part of the 9th exon sequence ...

Embodiment 2

[0063] Application of Fluorescence Quantitative PCR to Detect EGFRvIII

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Abstract

The present invention relates to a kit. Said kit can use cDNA obtained by means of reverse transcription of mRNA sample as template, and combines it with real-time fluorescent PCR detection technique, so that it can be mainly used for determining expression level of epidermal growth factor receptor III type mutant mRNA in various tumor tissues.

Description

technical field [0001] The invention belongs to the field of biological technology, and is a kit for accurately and quantitatively detecting the mRNA expression of human epidermal growth factor receptor type III mutant through real-time fluorescent quantitative PCR detection technology. Background technique [0002] Epidermal Growth Factor Receptor variant III (EGFRvIII) gene is derived from the in-frame deletion mutation at positions 276-1075 in the normal EGFR cDNA nucleotide sequence, corresponding to the 2nd to 7th outer positions of the mRNA sequence Deletion of exons. Its formation is caused by the rearrangement of the epidermal growth factor receptor (Epidermal Growth Factor Receptor, EGFR) gene amplification process, which has a certain relationship with the increase of EGFR mRNA expression. EGFRvIII can form dimers and self-phosphorylate independently of ligands such as EGF, which eventually leads to the constitutive activation of unregulated kinases, stimulates ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 杨蓓蓓曹江陈嘉张行
Owner ZHEJIANG UNIV
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