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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 expression level, gene expression, applied in the medical field

Inactive Publication Date: 2004-04-07
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

[0101] Isolate RNA from FPE tissue

[0102] RNA is extracted from paraffin-embedded tissue through the following general process.

[0103] A. Deparaffinization and hydration of sections

[0104] (1) Put a part of the about 10 μM slice in a 1.5 mL plastic centrifuge tube.

[0105] (2) Add 600 μL of xylene, and shake the mixture vigorously at room temperature (about 20-25° C.) for about 10 minutes.

[0106] (3) At room temperature, centrifuge the sample for about 7 minutes at the maximum speed of the bench top centrifuge (about 10-20,000xg).

[0107] (4) Repeat steps 2 and 3 until most of the paraffin is dissolved. Depending on the amount of paraffin contained in the original sample part, it usually needs to be repeated 2 or more times.

[0108] (5) Use lower alcohol, preferably 100% ethanol (about 600 μL) to vigorously shake for about 3 minutes to remove the xylene solution.

[0109] (6) Centrifuge the test tube for about 7 minutes according to step (3). Pour out and discard the sup...

Embodiment 2

[0123] mRNA reverse transcription and PCR

[0124] Reverse transcription: As exemplified in Example 1, RNA was isolated from microdissected or non-microdissected formalin-fixed paraffin-embedded (FPE) tissue, or RNA was based on the manufacturer’s Guidance, use QuickPrep TM MicromRNA purification kit (AmershamPharmaciaBiotech Inc., Piscataway, N.J.), separated from fresh or frozen tissues by a single-step guanidine isocyanate method. After ethanol precipitation and centrifugation, the RNA pellets were dissolved in 50 μL of 5 mM Tris / Cl pH 8.0. M-MLV reverse transcriptase can extend an oligonucleotide primer that hybridizes to a single-stranded RNA or DNA template in the presence of deoxynucleotides to produce a complementary strand. The resulting RNA was reverse transcribed using M-MLV reverse transcriptase from LifeTechnologies and random hexamers. Reverse transcription is performed by mixing 25 μL of RNA solution with 25.5 μL of "Reverse Transcription Mix" (see below). Place the...

Embodiment 3

[0130] Measure the uncorrected gene expression of EGFR (UGE)

[0131] Perform two parallel reactions. "Test" response and "calibration" response. Figure 7. The EGFR amplification reaction and the β-actin internal control amplification reaction are test reactions. The separate EGFR and β-actin amplification reactions are performed on the calibration RNA template and are called calibration reactions. The TaqMan® instrument produces 4 different cycle threshold (Ct) values: Ct of the test reaction EGFR And Ct β-肌动蛋白 , And the Ct of the calibration reaction EGFR And Ct β-肌动蛋白 . The Ct difference between the two reactions is determined according to the following formula:

[0132] ΔCt 试验 =Ct EGFR -Ct β-肌动蛋白 ("Test" response)

[0133] ΔCt 校准 =Ct EGFR -Ct β-肌动蛋白 ("Calibration" response)

[0134] The next step is to calculate the negative ΔCt power of 2 according to the following formula.

[0135] 2 -ΔCt试验 ("Test" response)

[0136] 2 -ΔCt校准 ("Calibration" response)

[0137] In order to obt...

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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-neumRNA, respectively.

Description

Invention field [0001] The present invention relates to a predictive method that is effectively used in medicine, especially cancer chemotherapy. More particularly, the present invention relates to assessing the survival ability of patients by analyzing the gene expression of tumor cells. In addition, the sensitivity of tumor cells to receptor tyrosine kinase-directed chemotherapy is determined by detecting the mRNA expression of human EGFR and HER2-neu genes. Background of the invention [0002] In Western countries, lung cancer is the leading cause of cancer-related deaths in both men and women. In the United States, approximately 171,000 new cases of lung cancer are diagnosed each year, and 160,000 cases die from this disease. In the past two decades, although some progress has been made in the detection and treatment of lung cancer, the overall 5-year survival is still less than 15%. Ginsberg et al., In: DeVita et al., Cancer: Principlesin Practiceof Oncology , Ed.5, pp.858-...

Claims

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Application Information

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