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Method of determining epidermal growth factor receptor and HER2-neu gene expression and correlation of levels thereof with survival rates

A technology of gene expression and oligonucleotide, applied in the field of determining the correlation between the gene expression of epidermal growth factor receptor and HER2-neu and its level and survival rate

Inactive Publication Date: 2011-10-19
RESPONSE GENETICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Likewise, there is no known method that provides reproducible measurements for the isolation of RNA from paraffin-embedded tissue samples

Method used

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  • Method of determining epidermal growth factor receptor and HER2-neu gene expression and correlation of levels thereof with survival rates
  • Method of determining epidermal growth factor receptor and HER2-neu gene expression and correlation of levels thereof with survival rates
  • Method of determining epidermal growth factor receptor and HER2-neu gene expression and correlation of levels thereof with survival rates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] RNA isolation from FPE tissue

[0104] RNA was extracted from paraffin-embedded tissues by the following general procedure.

[0105] A. Deparaffinization and Hydration of Sections

[0106] (1) Put a part of the slice at about 10 μM in a 1.5 mL plastic centrifuge tube.

[0107] (2) Add 600 μL of xylene, and shake the mixture vigorously at room temperature (about 20-25° C.) for about 10 minutes.

[0108] (3) At room temperature, centrifuge the sample for about 7 minutes at the maximum speed of the tabletop centrifuge (about 10-20,000 xg).

[0109] (4) Repeat steps 2 and 3 until most of the paraffin is dissolved. Depending on the amount of paraffin contained in the original sample portion, usually 2 or more replicates are required.

[0110] (5) Vigorously shake for about 3 minutes with lower alcohol, preferably 100% ethanol (about 600 μL), to remove the xylene solution.

[0111] (6) Centrifuge the test tube for about 7 minutes according to step (3). Decant and discar...

Embodiment 2

[0125] Reverse transcription and PCR of mRNA

[0126] Reverse transcription: RNA was isolated from microdissected or non-microdissected formalin-fixed paraffin-embedded (FPE) tissue as exemplified in Example 1, or RNA was isolated according to the manufacturer's guide, using QuickPrep TM MicromRNA purification kit (AmershamPharmaciaBiotechInc., Piscataway, N.J.), isolated from fresh or frozen tissue by a single-step guanidine isocyanate method. After ethanol precipitation and centrifugation, the RNA pellet was dissolved in 50 μL of 5 mM Tris / Cl, pH 8.0. M-MLV reverse transcriptase extends an oligonucleotide primer that hybridizes to a single-stranded RNA or DNA template in the presence of deoxynucleotides to generate a complementary strand. The resulting RNA was reverse transcribed using M-MLV reverse transcriptase from Life Technologies and random hexamers. Reverse transcription was performed by mixing 25 μL of RNA solution with 25.5 μL of "Reverse Transcription Mix" (see ...

Embodiment 3

[0132] Determination of Uncorrected Gene Expression (UGE) of EGFR

[0133] Two pairs of parallel reactions were performed. "Test" responses and "Calibration" responses. Figure 7 . The EGFR amplification reaction and the [beta]-actin internal control amplification reaction are experimental reactions. Separate EGFR and β-actin amplification reactions were performed on calibration RNA templates and were referred to as calibration reactions. The instrument generates 4 different cycle threshold (Ct) values: Ct of test response EGFR and Ct β-肌动蛋白 , and the Ct of the calibration response EGFR and Ct β-肌动蛋白 . The difference in Ct for the two reactions is determined according to the following formula:

[0134] ΔCt 试验 =Ct EGFR -Ct β-肌动蛋白 ("test" response)

[0135] ΔCt 校准 =Ct EGFR -Ct β-肌动蛋白 ("calibration" response)

[0136] The next step is to calculate 2 to the negative ΔCt power according to the following formula.

[0137] 2 -ΔCt试验 ("test" response)

[0138] 2 -ΔCt...

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Abstract

The present invention relates to prognostic methods which are useful in medicine, particularly cancer chemotherapy. The object of the invention to provide a method for assessing HER2-neu and / or EGFR expression levels in fixed or fixed and paraffin embedded tissues and prognosticate the probable sensitivity of a patient's tumor to treatment with receptor tyrosine kinase targeted chemotherapy by examination of the amount of HER2-neu and / or EGFR mRNA in a patient's tumor cells and comparing it to a predetermined threshold expression level for those genes. More specifically, the invention provides to oligonucleotide primer pairs EGFR and HER2-neu and methods comprising their use for detecting levels of EGFR and HER2-neu mRNA, respectively.

Description

[0001] The application date is November 9, 2001, the application number is 01822284.6, and the invention name is "method for determining the correlation between gene expression of epidermal growth factor receptor and HER2-neu and its level and survival rate" A divisional application of a patent application. field of invention [0002] The present invention relates to predictive methods useful in medicine, especially cancer chemotherapy. More particularly, the invention relates to assessing the viability of a patient by analyzing the gene expression of tumor cells. In addition, the sensitivity of tumor cells to receptor tyrosine kinase-directed chemotherapy regimens was determined by measuring the mRNA expression of human EGFR and HER2-neu genes. Background of the invention [0003] Lung cancer is the leading cause of cancer-related death in both men and women in Western countries. Each year in the United States, approximately 171,000 new cases of lung cancer are diagnosed ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/09
CPCC12Q1/6806C12Q1/6886C12Q2600/158A61P35/00C12Q2600/118
Inventor K·D·达南伯格
Owner RESPONSE GENETICS
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