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Detection of microRNAs in excreta as early diagnosis biomarker of lung cancer, colorectal cancer and bladder cancer

A colorectal cancer, excrement technology, applied in the field of medical diagnosis, can solve the problems of poor specificity and low sensitivity

Inactive Publication Date: 2013-09-25
SHENZHEN PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] At present, tumor markers and other non-invasive screening methods have low sensitivity and poor specificity. In order to solve this problem, the purpose of the present invention is to select tumor-specific miRNAs as new tumor markers; another purpose of the present invention It is to establish a simple, fast and economical miRNAs detection system; another purpose of the present invention is to combine the tumor miRNAs with high sensitivity and good specificity with this convenient detection system to establish a method for screening and early diagnosis Commercially valuable kits for tumors

Method used

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  • Detection of microRNAs in excreta as early diagnosis biomarker of lung cancer, colorectal cancer and bladder cancer
  • Detection of microRNAs in excreta as early diagnosis biomarker of lung cancer, colorectal cancer and bladder cancer
  • Detection of microRNAs in excreta as early diagnosis biomarker of lung cancer, colorectal cancer and bladder cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Tissue samples were collected and placed in cryopreservation tubes, placed in liquid nitrogen tanks, and then transferred to -80°C refrigerators for storage. Sputum samples were stored at 4°C after collection and processed within one week. Aliquot each sputum by 200μl into 1.5ml RNase free microcentrifuge tube, and add 400μl Sputolysin TM (0.1mg / ml, sigma) solution, vortex until the viscous sputum dissolves, incubate at 37°C for 30 minutes, and finally store the homogenized sputum at -20°C. After the stool samples were collected, 200 mg of each stool was divided into cryopreservation tubes and stored at -80°C. About 50ml of the urine sample was collected and centrifuged for 10 minutes, the supernatant was removed and the precipitate obtained was washed twice with PBS and frozen at -80°C. RNA was extracted from all specimens within one week after cryopreservation.

[0033] 2. Extraction of RNA

Embodiment 2

[0035] 1000μl Trizol TM (invitrogen) was added to the centrifuge tubes for storing specimens (including 25 mg of frozen tissue specimens ground into powder by liquid nitrogen grinding method, 200 μl of homogenized sputum, and frozen stool specimens by liquid nitrogen grinding method as described in Implementation 1. Grind into powder, 50ml of urine precipitate after centrifugation), mix by pipetting and then vortex for 20 seconds. Then add 200 μl chloroform and vortex mix for 20 seconds and let stand at room temperature for 5 minutes. The mixture was centrifuged at 4°C for 15 minutes to separate it into an aqueous phase and a red organic phase. Carefully transfer the aqueous phase (about 500 μl) to a clean 1.5ml centrifuge tube, add 4 μl of glycogen co-precipitation agent (Ambion) and 5000 μl of isopropanol, mix gently and place at room temperature for 20 minutes. Then centrifuge at 12000rpm for 10 minutes, centrifuge at 4°C for 15 minutes to precipitate the RNA, wash the pr...

Embodiment 3

[0040] Use of reverse transcription of miRNA MicroRNA Reverse Transcription Kit, Primers, and StepOnePlus TM The real-time quantitative PCR system (Applied Biosystems Inc.) was performed according to the instructions. The RT Master Mix for each reaction is configured as follows:

[0041]

[0042] Place RT Master Mix in a Nuclease-free PCR tube, mix gently and place on ice. Add 3.0 μl of miRNA primers and 5 μl of RNA extracted in Example 2 to each RT Master Mix, the final reaction system is 15 μl, and centrifuge at 1500 rpm for two minutes after mixing. Place on a PCR instrument at 16°C for 30 minutes, 42°C for 40 minutes, 85°C for 5 minutes, and 4°C for about 50 minutes, and store the generated product at -70°C.

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Abstract

The invention discloses a method for detection of microRNAs in excreta (sputum, faeces and urine) as a biomarker for early screening and diagnosing lung cancer, colorectal cancer and bladder cancer. The method comprises: (1) screening abnormal expression microRNAs existed in tissues of the lung cancer, the colorectal cancer and the bladder cancer; (2) by using resection tumor samples from patients of the lung cancer, the colorectal cancer and the bladder cancer, verifying the screened microRNAs in the tumor tissues; and (3) detecting expressions of the microRNAs in samples of sputum, faeces and urine from the patients of the lung cancer, the colorectal cancer and the bladder cancer, analyzing correlation with expression in the tissues, and confirming the microRNAs for early screening and diagnosis. The method is substantially characterized in that the microRNAs in the excreta is taken as the biomarker for early screening and diagnosing the cancers, and the method helps to realize noninvasive screening for the cancer patients and possesses a wide application scope.

Description

technical field [0001] The invention belongs to the technical field of medical diagnosis, and specifically relates to the screening of tumor-specific microRNAs, the establishment and optimization of a detection system for tumor-specific microRNAs, and its application in cancer screening and early diagnosis. Background technique [0002] Epithelial cancerous tissues include esophageal cancer, gastric cancer, pancreatic ductal cancer, colorectal cancer in the digestive system, lung cancer in the respiratory system, and bladder cancer in the urinary system. The mortality rate of these cancers is increasing year by year, and the screening and early diagnosis of high-risk groups can effectively reduce the mortality rate, but there is no ideal non-invasive screening method at present. microRNAs (miRNAs) are small non-coding RNAs that play a broad role in the regulation of gene expression. They are often abnormally expressed in the process of tumor formation, and special miRNAs exp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 李富荣余小舫王正齐晖费筠
Owner SHENZHEN PEOPLES HOSPITAL
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