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Gene set used for examination of colon cancer

A colorectal cancer and gene technology, applied in the field of screening colorectal cancer cells, can solve difficult problems

Active Publication Date: 2007-07-25
CANON KK +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is difficult to apply the method of targeting the mutation of DNA (K-RAS, P53, APC, etc.) in the body, and it is still in the research stage

Method used

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  • Gene set used for examination of colon cancer
  • Gene set used for examination of colon cancer
  • Gene set used for examination of colon cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 (Selection Step 1) The first selection of marker genes for colorectal cancer screening

[0048] (1) Extract total RNA

[0049] Peripheral blood, 6 cases of normal colorectal mucosa, 6 cases of early colorectal cancer tissues (Dukes A, B), and 19 cases of progressive colorectal cancer tissues (Dukes C, D) were collected, and total RNA was collected. The recovery of total RNA is carried out according to a conventional method as shown below.

[0050] First, each tissue is crushed (peripheral blood is used as it is), ISOGEN manufactured by NipponGene is added, homogenized, a small amount of chloroform is added, and centrifuged at 8000 rpm for 15 minutes. Obtain the supernatant, add the same amount of isopropanol as the obtained amount, incubate at room temperature for more than 15 minutes, centrifuge at 15000 rpm for 15 minutes, and recover the precipitate. And the total RNA was obtained by ethanol precipitation (70%).

[0051] (2) Obtain the expression profiles and s...

Embodiment 2

[0059] Example 2 Optimization of PCR conditions for 50 selected genes

[0060] (1) Reverse transcription (synthesis of the first strand)

[0061] Using the SUPER SCRIPT Choice System manufactured by Invitrogen, 5 μg of the total RNA of the progressive colorectal cancer tissue obtained in Example 1 was reverse transcribed with random hexanucleotide primers. Specifically, proceed as follows.

[0062] Adjust the total RNA to 10μg / 10μl, add 1μl of random hexanucleotide primer to it, and keep it at 68°C for 10 minutes to heat-denature it. After cooling quickly on ice for more than 2 minutes, add reagents as shown in Table 3, keep at 25°C for 10 minutes, 42°C for 60 minutes, and 68°C for 15 minutes. After cooling down quickly, and after spinning down, 1μl of RNase H was added and kept at 37°C for 20 minutes. After this, about 20 μl of the first-strand cDNA solution was recovered.

[0063] table 3

[0064] Reagent

Add amount

5×First chain buffer

4μl

10mM dNTP...

Embodiment 3

[0077] Example 3 (Selection Step 2) The second selection of 50 genes by RT-PCR

[0078] Almost none of the cells separated from the stool by MACS (Magnetic cell sorting) using epithelial cell-specific antibodies (Dynabeads Epithelial Enrich, Invitrogen International) (detailed in 1 of Example 6) Contains lymphocytes and hemoglobin. From this, it can be seen that the 50 genes selected in the selection step 1 are genes that can be effectively screened for colorectal cancer by the isolated cells. On the one hand, in order to directly extract RNA from the stool for screening, it is necessary to reselect genes that are completely undetectable in lymphocytes or red blood cells.

[0079] Therefore, as in Example 1, total RNA was extracted from the other 22 Dukes A and B cases, 8 Dukes C and D cases, and peripheral blood, and RT-PCR was performed using the following method to understand the characteristics of 50 genes.

[0080] (1) Reverse transcription reaction (synthesis of single-stran...

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Abstract

The invention provides the method which compared to conventional method, patients having colon cancer can be detected with higher accuracy. Colon cancer cells in a sample are screened by analyzing the amount of expression of at least 2 or more genes or products thereof selected from the group of genes listed in Tables 1 and 30.

Description

Technical field [0001] The present invention relates to a method for detecting early colorectal cancer. More specifically, it relates to a method for screening colorectal cancer cells using the expression level of a specific gene set in a sample (blood, stool, etc.) as an index, and primers, probes, and immobilized samples used in the method Wait. Background technique [0002] The disease that causes the largest number of cancer deaths in Japan is gastric cancer. However, in recent years, there has been a tendency for gastric cancer to decrease. Instead, colon cancer (colon cancer) has a tendency to increase significantly. The proportion of colorectal cancer in the total death toll has definitely increased year by year since 1995. It can be said that in the 21st century its death toll will replace gastric cancer and rank first. [0003] On the one hand, colorectal cancer worsens slowly. Even if it is a progressive cancer, if it can be completely curatively removed, the prognosis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/158C12Q2600/16
Inventor 石井美绘山本伸子川口正浩冈部哲夫佐佐木博己谷島聪松村保广松下尚之角田弘之原田邦男
Owner CANON KK
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