Application of miRNA, product of using same, and detection method thereof

A detection method and product technology, applied in the field of medical molecular biology, can solve the problems of no change in serum miRNA expression and less circulating miRNA in peripheral blood, and achieve the effect of good early diagnosis, promotion of early diagnosis and strong complementarity

Inactive Publication Date: 2018-06-15
QINGDAO UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

[0005] However, so far, there are few reports on the application of circulating miRNA in peripheral blood for the early diagnosis of acute myocardial infarction, and none of the existing early diagnostic markers for acute myocardial infarction is specific to the characteristics of changes in serum miRNA expression in the disease

Method used

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  • Application of miRNA, product of using same, and detection method thereof
  • Application of miRNA, product of using same, and detection method thereof
  • Application of miRNA, product of using same, and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1 sample processing

[0088] 1. 29 cases of patients with acute myocardial infarction and 21 cases of normal healthy controls received physical examination. 5mL peripheral blood was extracted using a coagulation tube, centrifuged in two steps at 4°C, and finally the upper serum was collected in an RNase / DNase-free centrifuge tube.

[0089] 2. Total RNA extraction from serum: Use TRI Reagent BD to extract RNA from serum:

[0090] (1) Lysis: 0.75mL TRI Reagent BD+0.25mL serum, mix vigorously;

[0091] (2) Internal reference: 50pM synthetic nematode cel-mir-39 was added to the lysate to provide internal control for validation studies;

[0092] (3) Two-phase separation: the above-mentioned lysate + 0.2mL chloroform, vigorously mixed and then allowed to stand for 10min, centrifuged at 12000g for 10min at 4°C;

[0093] (4) RNA precipitation: water layer + 0.6mL isopropanol + 3μL glycogen, precipitate for several hours;

[0094] (5) Wash RNA: 1 mL of 75% ethanol, c...

Embodiment 2

[0096] Example 2 Real-time PCR detection

[0097] miR-17-5p and miR-126-5p in serum were detected using miRNA detection kits (including reverse transcription kits, specific miRNA primers and probes, fluorescent quantitative detection kits, etc.) and the levels of miR-145-3p and the reference miRNA (cel-mir-39), the average value of the three results (Ct values) was used as the Ct value of the specific miRNA.

Embodiment 3

[0098] The standardized processing of embodiment 3 data

[0099] According to the measured Ct values ​​of miR-17-5p, miR-126-5p, miR-145-3p and reference miRNA (cel-mir-39) in the sample serum. With cel-mir-39 as a reference, the relative content of specific miRNA in serum was obtained, and the classic 2 in PCR detection －ΔCt The way of represents the level of specific miRNA in serum (ΔCt is the difference between the Ct value of target miRNA and reference cel-mir-39). The result is as Figure 1 to Figure 3 and Figure 4A to Figure 7B shown. From figure 1 It can be seen that the expression level of circulating miR-17-5p in patients with acute myocardial infarction before and after PCI is significantly higher than that in healthy controls, indicating that miR-17-5p can be developed as a diagnostic marker. From figure 2 It can be seen that the expression level of circulating miR-126-5p in patients with acute myocardial infarction before and after PCI is significantly high...

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Abstract

The invention provides an application of miRNA, a product of using the same, and a detection method thereof, and relates to the technical field of medical molecular biology. The invention provides a miR-17-5p, miR-126-5p or miR-145-3p marker set taken as a marker for assisting the early diagnosis of heart diseases. The matured marker can stably exist in cytoplasm, thus, in acute myocardial infarction, miRNA in serum is released and enters a blood circulation system before structural proteins, and people can detect the level of the miRNA to assist the early diagnosis of acute myocardial infarction. Selected three markers are strongly complementary, and can all be taken as the markers for the early diagnosis of heart diseases. After three markers are used together, the precision is improved,and the sensitivity and specificity of disease diagnosis are greatly improved.

Description

technical field [0001] The invention relates to the technical field of medical molecular biology, in particular to an application of miRNA, a product using the same and a detection method. Background technique [0002] Acute myocardial infarction (AMI) is myocardial necrosis caused by acute and persistent coronary ischemia and hypoxia. It is one of the main causes of morbidity and mortality worldwide and has seriously endangered human health. According to WHO statistics, acute myocardial infarction It has become the number one cause of death worldwide. The incidence of acute myocardial infarction in my country is about 55 / 100,000, and the incidence has been on the rise in recent years. Acute myocardial infarction is a complex and critical disease involving multiple etiologies and multiple physiological, biochemical and pathological changes. At present, the methods of early reperfusion treatment of acute myocardial infarction, such as emergency thrombolysis or early interve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/178
Inventor 李培峰薛升刘大成周长勇张蕾张媛丁晗王玉亓洪昭
Owner QINGDAO UNIV
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