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Detection and application of a novel molecular marker hsa_circ_0074362 for gastric cancer

A gastric cancer and circular technology, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of lack of early diagnosis methods, unobvious early symptoms of gastric cancer, and insufficient sensitivity and specificity of gastric cancer. advanced questions

Active Publication Date: 2019-11-22
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 early diagnosis methods [di Mario F et al. -9. The sensitivity and specificity of CA50 and CA125 in gastric cancer are not high, and CA72-4, which has high specificity for gastric cancer, is usually increased in stage III-IV of gastric cancer

Method used

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  • Detection and application of a novel molecular marker hsa_circ_0074362 for gastric cancer
  • Detection and application of a novel molecular marker hsa_circ_0074362 for gastric cancer
  • Detection and application of a novel molecular marker hsa_circ_0074362 for gastric cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Detection of the expression of hsa_circ_0074362 in gastric cancer tissue and normal gastric tissue:

[0035] 1. Microarray analysis: Human circRNA Array (6×7K) microarray from Arraystar Company of the United States was used to detect the level of circRNA in gastric cancer tissue and normal tissue.

[0036] 2. Chip result: the result is as follows figure 1 And shown in Table 1.

[0037]Table 1 Typical circRNAs differentially expressed in gastric cancer tissues (P<0.05)

[0038]

[0039] 3. Analysis of results: The difference between hsa_circ_0074362 in gastric cancer tissue and normal tissue was 7.75 times, suggesting that hsa_circ_0074362 may function as a tumor suppressor gene in gastric cancer.

Embodiment 2

[0040] Embodiment 2: Collect normal gastric tissue as a normal control group, and perform circular RNA detection according to the following steps, including the following steps:

[0041] a. Tissue collection: Gastric cancer tissues were collected in sterile centrifuge tubes added with RNA preservation solution, and stored in a -80°C ultra-low temperature refrigerator when not in use;

[0042] b. Release RNA: Weigh about 20 mg of tissue into a 2 mL centrifuge tube, place on ice and add 1 mL of TRIzol. Use a hand-held automatic homogenizer to fully homogenize the tissue until there is no solid, and let it stand at room temperature for 10 minutes to fully release the RNA in the tissue into the solution;

[0043] c. Chloroform extraction: Add 0.2mL of chloroform, vortex and mix well, let stand at room temperature for 10 minutes; centrifuge at 13,000rpm for 10 minutes at 4°C, at this time, the liquid is separated, and RNA is enriched in the upper aqueous phase, be careful Pipette ...

Embodiment 3

[0053] Example 3 Method for Gastric Cancer Detection Using the hsa_circ_0074362 Biomarker

[0054] Steps include:

[0055] 1. Collect tissue samples;

[0056] 2. Extraction of circular RNA in gastric cancer tissue (the extraction method is the same as in Example 2);

[0057] 3. The reverse transcription and fluorescence quantitative PCR reactions were performed as in "reverse transcription and fluorescence quantitative reactions" in Example 2;

[0058] 4. Using hsa_circ_0074362 as a biomarker for gastric cancer detection, the expression levels of hsa_circ_00074362 in cancer tissues and adjacent tissues of 127 gastric cancer patients were analyzed. The ΔCt of hsa_circ_00074362 in the gastric cancer patient group was significantly higher than that in the normal group, P image 3 shown. The cut-off value of hsa_circ_00074362 as a gastric cancer marker is 12.17. When the PCR relative quantitative value ΔCt of the hsa_circ_0074362 biomarker in the sample is less than or equal to ...

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Abstract

The invention relates to a cyclic RNA molecular marker for gastric cancer diagnosis. The molecular marker is characterized in that the cyclic RNA (ribonucleic acid) is hsa-circ-0074362. The invention also provides a method for detecting the cyclic RNA molecular marker for gastric cancer. The method comprises the following steps: (1) collecting gastric cancer tissues; (2) obtaining RNA with high concentration and purity by using a drying and precipitating method; (3) performing reverse transcription to produce cDNA; (4) performing real-time quantitative PCR (Polymerase Chain Reaction) detection by fluorescent dyes; (5) counting Ct values of target cyclic RNA and house-keeping genes in the gastric cancer tissues; (6) performing relative quantification on the cyclic RNA by adopting a calculation method of Delta Ct=Ct (target circRNA)-Ct(reference). The method provided by the invention is convenient to operate and economic, and the product is high in purity and concentration. Compared with the prior art, the method provided by the invention has the advantages that specific down-regulated expression hsa-circ-0074362 in the gastric cancer tissues can serve as a novel molecular marker for gastric cancer diagnosis.

Description

technical field [0001] The invention relates to a method for detecting circular RNA in gastric cancer tissue, in particular to a method for detecting circular RNA in gastric cancer tissue by real-time quantitative RT-PCR using a fluorescent dye method and an application thereof. Background technique [0002] Gastric cancer is one of the most common malignant tumors in the world, with about 1 million new cases every year. The death rate of gastric cancer in men is the second among all kinds of tumors, and it is the fourth in women [Chen W, Zheng R, Zeng H, Zhang S, HeJ. Annual report on status of cancer in China, 2011. Chin J Cancer Res, 2015, 27(1):2-12.]. And my country is a high-incidence area of ​​gastric cancer. Every year, 400,000 gastric cancer patients are newly discovered in my country, accounting for about 42% of the world's gastric cancer incidence. Due to the lack of obvious early symptoms of gastric cancer and the lack of specific early diagnosis methods [di Mar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/113
CPCC12Q1/6886C12Q2600/158
Inventor 郭俊明谢依肖丙秀
Owner NINGBO UNIV