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Method for early detection on colorectal cancer on basis of NDRG4 gene methylation sequence

A technology for early detection of colorectal cancer, applied in the field of biotechnology and disease detection, can solve problems such as inactivation of tumor suppressor genes

Active Publication Date: 2018-10-19
HANGZHOU NEW HORIZON HEALTH TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Hypermethylation of CpG islands in gene promoter regions is associated with gene silencing and may inactivate tumor suppressor genes

Method used

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  • Method for early detection on colorectal cancer on basis of NDRG4 gene methylation sequence
  • Method for early detection on colorectal cancer on basis of NDRG4 gene methylation sequence
  • Method for early detection on colorectal cancer on basis of NDRG4 gene methylation sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0389] Example 1. Using TaKaRa MiniBEST FFPE DNA Extraction Kit to Extract Genomic DNA from FFPE Samples

[0390] Specific steps are as follows:

[0391] Use a sterilized scalpel to scrape 30 mg of paraffin section tissue to remove excess paraffin;

[0392] Put the paraffin section tissue into a 1.5mL centrifuge tube, add 500μL Buffer DP, mix well, place in a water bath at 80°C for 1 minute, vortex for 10 seconds while hot, add 180μL Buffer GL, and vortex;

[0393] Then centrifuge at room temperature at 12000rpm for 1 minute, the solution forms two layers, the upper oil phase, the lower water phase, add 20 μL Proteinase K, 20 mg / mL and 10 μL RNase, 10 mg / mL to the lower water phase, pipette to mix, and then 56 ° C water bath After 1 hour, place the treated sample in a 90°C water bath for 30 minutes, and cool to room temperature;

[0394] Add 200 μL Buffer GB and 200 μL 100% ethanol to the processed sample, and vortex for 10 seconds.

[0395] Then centrifuge at room temperat...

Embodiment 2

[0405] Example 2. Genomic DNA Extraction from Feces Samples

[0406] Take a stool sample (4-6g) and add 40mL of lysate, vortex, mix thoroughly and incubate at 50°C for 16 hours;

[0407] After incubation, centrifuge at 5000rpm for 10min. Pay attention to weighing and balance before centrifugation. After centrifugation, carefully take out the centrifuge tube and do not shake violently;

[0408] Pipette 9 mL of supernatant into a new 50 mL centrifuge tube, then add 1 mL of extraction auxiliary solution, 60 μL of magnetic bead solution, and 10 mL of isopropanol, vortex for 10 seconds, incubate at 65°C for 20 minutes, and mix up and down every 5 minutes during the incubation period. Even once;

[0409] After the incubation, place the 50mL centrifuge tube on the magnetic stand and let it stand for 3 minutes. After the magnetic beads are fully adsorbed on the tube wall, discard the waste liquid;

[0410] Take the centrifuge tube out of the magnetic stand, add 12mL washing solution...

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Abstract

The invention discloses a method for early detection on a colorectal cancer on the basis of a NDRG4 gene methylation sequence. The auxiliary diagnosis is performed on the early colorectal cancer through detecting whether a section of methylation sequence of a NDRG4 gene promoter region is methylated or not; the method comprises the following steps: extracting DNA from tissue and fecal samples, treating with hydrosulfite, then performing real-time quantitative PCR determination and judging a methylation level through calculating whether an amplified Ct ratio difference value between a target gene NDRG4 and an internal reference gene B2M is within a decision critical value or not. The method is noninvasive and can be used for the auxiliary diagnosis of the early colorectal cancer.

Description

technical field [0001] The invention relates to the fields of biotechnology and disease detection, in particular to an early detection method for colorectal cancer based on the NDRG4 gene methylation sequence. Background technique [0002] Colorectal cancer (CRC) is the fourth most common cancer in men and the third most common cancer in women in the world, with the highest incidence in developed countries. CRC affects approximately 5% of the U.S. population and ranks second in cancer-related mortality. In recent years, with the improvement of people's living standards in our country, the change of living habits and dietary structure, and the increasingly serious environmental pollution, the incidence of colorectal cancer in our country has been increasing. According to the 2013 research report of the National Cancer Early Diagnosis and Early Treatment Project Expert Committee, the rate of advanced adenoma and early bowel cancer among high-risk groups over the age of 40 in ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6851C12Q1/6886C12Q2600/154C12Q2523/125C12Q2531/113C12Q2545/101C12Q2545/113
Inventor 李慧李存耀郑伟贤杨蛟刘刚吕宁陈一友
Owner HANGZHOU NEW HORIZON HEALTH TECH CO LTD
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