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Detection and application of novel gastric cancer molecular marker tRF-5026a

A technology of trf-5026a and molecular markers, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of short survival time, low detection rate of gastric cancer, and no early symptoms of gastric cancer. Obvious issues

Active Publication Date: 2018-06-15
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of obvious early symptoms of gastric cancer and the lack of specific diagnostic methods for early gastric cancer [Liu X, Meltzer SJ. Gastric Cancer in the Era of Precision Medicine. Cell Mol Gastroenterol Hepatol, 2017,3(3):348-358], there have been The detection rates of tumor markers such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (Carbohydrate antigen 19-9, CA19-9) in gastric cancer are not high
The vast majority of gastric cancer patients are diagnosed at an advanced stage, which makes the survival period of patients shorter [Chia NY, Tan P. Molecular classification of gastric cancer. Ann Oncol, 2016, 27(5): 763-9.]

Method used

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  • Detection and application of novel gastric cancer molecular marker tRF-5026a
  • Detection and application of novel gastric cancer molecular marker tRF-5026a
  • Detection and application of novel gastric cancer molecular marker tRF-5026a

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Detection of the expression of tRF-5026a in gastric cancer tissue and normal gastric tissue:

[0035] 1. Sequencing detection: The tRF single-end sequencing reagent of American Arraystar Company was used to detect the level of tRF in gastric cancer tissues and normal tissues.

[0036] 2. Analysis of results: The difference of tRF-5026a between gastric cancer tissue and normal tissue was 4.83 times, suggesting that tRF-5026a may function as a tumor suppressor gene in gastric cancer.

Embodiment 2

[0037] Embodiment 2: Collect normal gastric tissue as a normal control group, and perform tRF detection according to the following steps, including the following steps:

[0038] Step a. Collect gastric cancer tissue samples: take 10-20 mg of gastric cancer tissue and soak in 1-2 mL of RNA preservation solution in a nuclease-free centrifuge tube;

[0039]Step b, lyse tissue: Add 1-2 mL of total RNA extraction reagent TRIzol to the centrifuge tube in step a, fully grind and homogenize the gastric cancer tissue into a homogenate, and let it stand at room temperature for 5-8 minutes to fully absorb the RNA in the tissue. released into the homogenate;

[0040] Step c, Chloroform extraction: Add 200-300 μL of chloroform, vortex and shake, and let stand at room temperature for 3 minutes; centrifuge at 12,000 rpm and 4°C for 15 minutes. At this time, the liquid will be separated, and the RNA is rich in the upper aqueous phase Collect, carefully pipette about 0.5mL-1.5mL of the upper ...

Embodiment 3

[0059] Method for gastric cancer detection using tRF-5026a biomarker

[0060] Steps include:

[0061] 1. Collect tissue samples;

[0062] 2. Extraction of RNA in gastric cancer tissue (extraction method is the same as in Example 2);

[0063] 3. The pre-treatment reverse transcription and fluorescent quantitative PCR reactions are operated as in the "pre-treatment reverse transcription and fluorescent quantitative reaction" in Example 2;

[0064] 4. Using tRF-5026a as a biomarker for gastric cancer detection, the expression levels of tRF-5026a in cancer tissues and adjacent tissues of 86 gastric cancer patients were analyzed. The ΔCq of tRF-5026a in the gastric cancer patient group was significantly higher than that in the normal group, P image 3 shown. The cut-off value of tRF-5026a as a gastric cancer marker is 14.03. When the PCR relative quantitative value ΔCq of tRF-5026a biomarker in the sample is less than or equal to 14.03, it is considered as a non-gastric cancer sa...

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Abstract

The invention relates to a tRF molecular marker for gastric cancer diagnosis. The tRF molecular marker for the gastric cancer diagnosis is characterized in that the tRF is tRF-5026a. The invention further provides a method for detecting the gastric cancer tRF molecular marker. The method comprises the following steps: (1), collecting a gastric cancer tissue; (2), acquiring RNA with relatively highconcentration and purity; (3), pretreating and reversely transcribing into cDNA; (4), performing qRT-PCR detection by a fluorescent dye method; (5), using specific upstream and downstream amplification primers by the tRF and an external reference gene; (6), through statistics, acquiring Cq values of the target tRF and the external reference gene in the gastric cancer tissue; (7), relatively quantifying the tRF by a calculation method that a delatCq is equal to Cq (target tRF)-Cq (reference). Compared with the prior art, the invention has the advantage as follows: through specific expression downregulation in the gastric cancer tissue, the tRF-5026a can be used as a novel molecular marker for the gastric cancer diagnosis.

Description

technical field [0001] The invention relates to a method for detecting tRNA-derived fragments in gastric cancer tissue, in particular to a method for detecting tRF in gastric cancer tissue by real-time quantitative reverse transcription-polymerase chain reaction using a fluorescent dye method and its application. Background technique [0002] In global cancer statistics, the incidence of gastric cancer ranks first, and China is a country with a high incidence of gastric cancer. Despite the continuous improvement of diagnostic technology and the increasing variety of comprehensive treatment methods based on surgical treatment, gastric cancer is still one of the leading cancer-death diseases in the world, with about 1 million new cases and more than 720,000 deaths every year [Zhao Qian, Yang Xiaoyan, Gan Runliang. Advances in molecular typing of gastric cancer. Chinese Journal of Pathology, 2016,45(10):737-741]. China is a country with a high incidence of gastric cancer. Ther...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/158
Inventor 郭俊明谢依肖丙秀
Owner NINGBO UNIV