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Liver cancer biomarkers

A technology for biomarkers and products, which can be used in sugar derivatives, biochemical equipment and methods, and the determination/inspection of microorganisms, and can solve problems such as easy bleeding

Inactive Publication Date: 2007-09-19
GENENEWS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in about 0.4% of cases, the patient bleeds fatally as a result of the biopsy because some tumors are heavily vascular and bleed very easily

Method used

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  • Liver cancer biomarkers
  • Liver cancer biomarkers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0606] RNA isolation from lysed blood

[0607] 10 ml of whole blood was collected in a Vacutainer and centrifuged at 2,000 rpm for 5 minutes at 4°C to remove the plasma layer. Add lysis buffer at a ratio of 3 parts lysis buffer to 1 part blood (lysis buffer (1 L): 0.6 g EDTA; 1.0 g KHCO 2 , 8.2g NH 4 Cl, adjusted to pH 7.4 with NaOH). Mixed samples were placed on ice for 5-10 minutes until clear. The lysed samples were centrifuged at 1000 rpm for 10 min at 4°C and the supernatant was discarded by aspiration. The pellet was resuspended in 5 ml of lysis buffer and the lysed sample was centrifuged again at 1000 rpm for 10 min at 4°C. The cells were homogenized with TRIzol_(GIBCO / BRL) at a ratio of approximately 6 ml TRIzol_ per 10 ml of original blood sample and shaken well. The samples were left to stand at room temperature for 5 minutes. RNA was extracted with 1.2ml chloroform / 1ml TRIzol®. Centrifuge the samples at 12,000 x g for 5 min at 4 °C and collect the upper layer...

Embodiment 2

[0609] RNA isolation from whole blood

[0610] 100 μl of whole blood was collected in a microcentrifuge tube, centrifuged at 2,000 rpm (800 g) at 4° C. for 5 minutes, and the supernatant was removed. The cells were homogenized with TRIzol (GIBCO / BRL) at a ratio of approximately 6 μl TRIzol per 10 μl of original blood sample and shaken thoroughly. The samples were left to stand at room temperature for 5 minutes. RNA was extracted with 12 μl chloroform / 10 μl TRIzol. Centrifuge the samples at 12,000 x g for 5 min at 4 °C and collect the upper layer. Add isopropanol to the upper layer at a ratio of 5 μl / 10 μl TRIzol®. Samples were left at -20°C overnight or at -20°C for 1 hour. Precipitate RNA according to known methods, air dry the RNA pellet, and resuspend the pellet in DEPC-treated ddH 2 O middle. RNA samples can also be stored in 75% ethanol, under which conditions the samples are stable at room temperature for shipping.

[0611] RNA isolation from centrifuged lysed blo...

Embodiment 3

[0616] Target nucleic acid preparation and hybridization

[0617] Preparation of fluorescent DNA probes from mRNA

[0618] Fluorescently labeled target nucleic acid samples of RNA are prepared for analysis with the arrays of the invention.

[0619] 1 μg of Oligo-dT primer was annealed to 10 μg of total RNA in a total volume of 10 μl obtained from the blood of patients diagnosed or suspected of having liver cancer by heating at 70 °C for 10 min and cooling on ice. separate. Incubate samples at 42°C for 40 minutes to reverse transcribe mRNA in a 25 μl volume containing final concentrations of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 25 mM DTT, 25 mM unlabeled dNTPs, 400 units of Superscript II (200 U / μl, BRL) and 15 mM Cy3 or Cy5 (Amersham). The reaction was stopped by adding 2.5 μl of 55500 mM EDTA and 5 μl of 1 M NaOH and incubating at 65° C. for 10 minutes. The reaction mixture was neutralized by adding 12.5 μl of 1M TrisHCl (pH 7.6).

[0620] Labeled target nuc...

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Abstract

The invention relates to the identification and selection of biomarkers which demonstrate particular advantage in identifying individuals having liver cancer. The invention further relates to useful combinations of biomarkers for diagnosing liver cancer. The invention further provides for the polynucleotides and polypeptides and kits thereof for use as a tool to diagnose disease and to monitor the efficacy of therapeutic regimens. The invention further provides a method of selecting biomarker combinations and the combinations thus identified for diagnosis of liver cancer. Also encompassed by the invention are screening methods to identify therapeutic targets for treating liver cancer, and identify single nucleotide point mutations related to liver cancer.

Description

[0001] This application claims priority to US Provisional Application No. 60 / 516,853, filed November 3,2003. The entire teachings of the above applications are incorporated herein by reference. 1. Field of invention [0002] The present invention relates to the identification and selection of new biomarkers differentially expressed in liver cancer and combinations of new biomarkers differentially expressed in liver cancer, and methods of selecting said new biomarker combinations. The present invention also relates to the use of the polynucleotide and / or protein specifically hybridized with the product of the biomarker of the present invention to diagnose liver cancer and its kit. The invention also relates to a screening method for monitoring the effect of a treatment plan, identifying a therapeutic target for treating liver cancer, and identifying single nucleotide point mutations related to liver cancer. 2. Background of the invention [0003] Liver cancer is a cancer wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C12Q1/68A61K39/395C07K14/00C07K16/30C12NG01N33/574
CPCC07K14/00C07K16/303
Inventor 刘宗正汤姆·亚格亚当·登普西塞缪尔·尚
Owner GENENEWS
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