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Detection and application of new molecular marker hsa-circ-0001017 of gastric cancer

A technology of molecular markers and gastric cancer, applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of low sensitivity and specificity, and achieve convenient and fast sampling, fast detection, The effect of high sensitivity

Active Publication Date: 2017-04-26
NINGBO UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the sensitivity and specificity of the existing tumor markers CEA, CA19-9, CA50, and CA125 in the diagnosis of gastric cancer are not high. Although CA72-4 has high specificity for gastric cancer, it is usually only found in gastric cancer Increased in stage III-IV

Method used

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  • Detection and application of new molecular marker hsa-circ-0001017 of gastric cancer
  • Detection and application of new molecular marker hsa-circ-0001017 of gastric cancer
  • Detection and application of new molecular marker hsa-circ-0001017 of gastric cancer

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1: Detecting the expression of hsa_circ_0001017 in the plasma of gastric cancer patients and healthy people:

[0034] 1. Chip analysis: Human circRNAArray (6×7K) chips from Arraystar, USA were used to detect the level of circRNA in the plasma of gastric cancer patients and healthy people.

[0035] 2. Chip result: the result is as follows figure 1 And shown in Table 1.

[0036] Table 1 list of significant differences

[0037] Table 1 Typical circRNAs differentially expressed in the blood of patients with gastric cancer (P<0.05)

[0038]

[0039] 3. Analysis of the results: The difference between hsa_circ_0001017 in the plasma of gastric cancer patients and healthy people was 6.85 times, suggesting that hsa_circ_0001017 may function as an oncogene in gastric cancer.

Embodiment 2

[0040] Embodiment 2: The plasma of healthy people was collected as a normal control group, and each part of blood was tested for circular RNA according to the following steps, including the following steps:

[0041] a. Plasma collection: Anticoagulate whole blood with EDTA, centrifuge at 3000 rpm for 10 minutes at room temperature, take about 300 μL of light yellow plasma from the upper layer, transfer it to an RNase-free centrifuge tube, and store it on ice;

[0042] b. Release RNA: Add 800 μL of TRIzol-LS (commercially available) to the plasma to make up to a total volume of 1 mL, vortex and oscillate to mix, and let stand at room temperature for 15 minutes to fully release the RNA in the plasma;

[0043]c. Chloroform extraction: Add 40 μl of chloroform / isoamyl alcohol solution (ratio: 24:2), shake it up and down by hand until it becomes milky, and let it stand at room temperature for 5 minutes. At this time, the initial layering of the solution can be seen. The upper layer ...

Embodiment 3

[0055] The preoperative plasma and the 10th day postoperative plasma were collected from patients with gastric cancer for detection, and the method was basically the same as that in Example 1. Depend on image 3 It can be seen that there are a large number of positive and negative droplets at the "." point, and the distribution is very distinct, and they are concentrated above and below the threshold. The fluorescent signal value of the positive droplets after surgery decreases. The copy number of circular RNA hsa_circ_0001017 is 4100copies / mL, which is higher than the average copy number of 1070copies / mL in normal human plasma samples, while the copy number of postoperative plasma circular RNA hsa_circ_0001017 is 600copies / mL, indicating that the circular RNA of gastric cancer patients before and after surgery The copy number of the similar RNA hsa_circ_0001017 was significantly decreased to prove that hsa_circ_0001017 can be sensitively used as a molecular marker for the dia...

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Abstract

The invention relates to a cyclic RNA molecular marker for diagnosis of gastric cancer, the cyclic RNA molecular marker is characterized in that the cyclic RNA is hsa-circ-0001017, the invention also provides a method for detection of the cyclic RNA molecular marker in plasma, and the method comprises the following steps: (1) collecting blood, and extracting total RNA in the plasma; (2) performing reverse transcription of the total RNA into cDNA; (3) performing droplet digital PCR detection of a cDNA solution of the step (2) by use of specific amplification back-to-back primers and amplification upstream and downstream primers of housekeeping gene GAPDH, after the completion of the reaction, detecting fluorescence signal values of all droplets, setting a threshold, and determining whether the droplets include the cyclic RNA or the housekeeping gene GAPDH, wherein the droplets higher than the threshold are positive droplets, and the droplets below the threshold are negative droplets; and (4) counting the number of the positive droplets, and calculating the copy number of the hsa-circ-0001017 and the housekeeping gene GAPDH in the plasma for quantitative detection of the hsa-circ-0001017 and the housekeeping gene GAPDH in the plasma. Compared with the prior art, the advantages are that the hsa-circ-0001017 can be specifically expressed in plasma in patients with gastric cancer, and can be used as a new molecular marker for diagnosis of the gastric cancer.

Description

technical field [0001] The invention relates to a method for detecting circular RNA in plasma, in particular to a method for detecting circular RNA in plasma by droplet digital PCR 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 techniques and the increasing availability of comprehensive treatment methods based on surgical treatment, gastric cancer is still one of the leading cancer-causing diseases in the world. Moreover, the sensitivity and specificity of the existing tumor markers CEA, CA19-9, CA50, and CA125 in the diagnosis of gastric cancer are not high. Although CA72-4 has high specificity for gastric cancer, it is usually only found in gastric cancer The increase occurred only in stages III-IV. Therefore, it is imperative to find reliable biomarkers, such as lncRNAs, as early di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/158C12Q2600/178
Inventor 郭俊明李田文肖丙秀
Owner NINGBO UNIV
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