Exosome miRNA marker of rheumatoid arthritis and kit

A rheumatoid and marker technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, organic compound library, etc., can solve the problems of easy degradation or denaturation, errors, low diagnostic sensitivity and specificity of rheumatoid arthritis, etc. Achieve high sensitivity and specificity, respond well to disease progression, and ensure stability

Active Publication Date: 2018-12-21
SUZHOU UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

Traditional biochemical markers such as rheumatoid factor and anti-cyclic citrullinated peptide antibody are difficult to balance sensitivity and specificity in the diagnosis of rheumatoid arthritis, so the development of new markers of rheumatoid arthritis is conducive to early diagnosis and prevention of the disease , and then carry out effective intervention to reduce the harm of rheumatoid arthritis to the mechanism
[0003] At present, most of the diagnostic markers of rheumatoid arthritis adopt ELISA or electrochemiluminescence method to detect rheumatoid arthritis-related proteins in serum, and there are mainly the following problems: 1) some rheumatoid arthritis markers (such as cells factor), ELISA cannot detect; 2) free rheumatoid arthritis markers in plasma are easily degraded or denatured due to storage conditions and detection time, which will affect the detection results and cause errors; 3) rheumatoid arthritis in plasma Markers (eg, rheumatoid factor, erythrocyte sedimentation rate), low diagnostic sensitivity and specificity for rheumatoid arthritis
Currently there is no use of exosomal miRNAs for the diagnosis of rheumatoid arthritis

Method used

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  • Exosome miRNA marker of rheumatoid arthritis and kit
  • Exosome miRNA marker of rheumatoid arthritis and kit
  • Exosome miRNA marker of rheumatoid arthritis and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Microarray screening experiment of differentially expressed microRNA in plasma exosomes

[0031] 1. After obtaining the informed consent of the patients, the peripheral blood samples of 9 patients diagnosed with rheumatoid arthritis and 9 healthy people as normal controls were collected from the First People's Hospital Affiliated to Soochow University and anticoagulated with sodium citrate. Tube. 3 people make a group, a total of 6 groups.

[0032] 2. Exosome extraction. The exoRNeasy Serum / Plasma kit from Qiagen was used to extract plasma exosome miRNA. The kit uses a spin column method to extract exosomes, which is divided into two steps: the separation of plasma exosomes and the extraction of total exosome RNA. . First, take 1ml of plasma, filter it with a 0.22μm needle filter, mix it with an equal volume of Buffer XBP, and then filter it through a spin column to fully combine the exosomes with the spin column, wash it with Buffer XWP, and finally pass i...

Embodiment 2

[0036] Example 2: RT-PCR experiment of plasma exosome miR-204-5p

[0037] 1. According to the miRNA microarray results, 9 miRNAs were selected from 14 differentially expressed miRNAs according to the following conditions for RT-PCR verification. The specific conditions are: (1) the miRNA has not been reported before and has been verified by RT-PCR in the RA population; (2) the miRNA has a high FC value; (3) the miRNA has a high expression in the chip Level. The primers of selected miRNAs such as miR-204-5p are shown in Table 1. For the RT-PCR detection of candidate miRNAs in the plasma exosomes of patients with rheumatoid arthritis and normal controls, strict quality control was implemented throughout the study, and each sample was continuously tested at least three times.

[0038] Table 1 miR-204-5p primer sequence list

[0039]

[0040] 2. After obtaining informed consent from patients, two independent batches of samples were collected from the First People's Hospital ...

Embodiment 3

[0051] Example 3: Analysis of the number of amplification cycles and clinical indicators of miR-204-5p

[0052] The clinical characteristics of the selected patients are shown in Table 2. Using Pearson correlation, the amplification cycle number (CT value) and clinical indicators of plasma exosomal miR-204-5p were analyzed, and the results are shown in Table 3. The results showed that the correlations between plasma exosomal miR-204-5p amplification cycles and erythrocyte sedimentation rate (ESR), rheumatoid arthritis activity (DAS28), and C-reactive protein (CRP) were 0.394, 0.336, and 0.471, respectively. There is no necessary relationship with rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP). Figure 4 The ordinate is the amplification cycle number (CT value) of plasma exosomal miR-204-5p, and the abscissa is the disease activity of rheumatoid patients, and the correlation between the two is 0.336.

[0053] Table 2 Clinical information of patients with r...

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Abstract

The invention discloses an exosome miRNA marker of rheumatoid arthritis and a kit and belongs to the technical field of biological detection. According to the exosome miRNA marker of rheumatoid arthritis, an exosome miR-204-5p sequence and a corresponding primer can be used for preparing a rheumatoid arthritis diagnostic kit; very high sensitivity and specificity are realized; and the exosome miRNA marker of the rheumatoid arthritis is beneficial for promoting the developments of early diagnosis, treatment predication and recurrence monitoring of the rheumatoid arthritis in China.

Description

technical field [0001] The invention relates to an exosome miRNA marker and a kit for rheumatoid arthritis, belonging to the technical field of biological detection. Background technique [0002] Rheumatoid arthritis is a systemic autoimmune disease characterized by symmetric, erosive synovitis leading to destruction of bone or cartilage. Rheumatoid arthritis brings great suffering to patients, reduces the quality of life of patients, has a high late disability rate, and shortens life expectancy. Rheumatoid arthritis has a long treatment cycle and is prone to recurrence. At present, there are no effective preventive and radical measures. Studies have shown that early intervention in rheumatoid arthritis can effectively control the disease. Traditional biochemical markers such as rheumatoid factor and anti-cyclic citrullinated peptide antibody are difficult to balance sensitivity and specificity in the diagnosis of rheumatoid arthritis, so the development of new markers of r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11C40B40/06
CPCC12Q1/6883C12Q2600/158C40B40/06
Inventor 雷署丰武龙飞邓飞艳
Owner SUZHOU UNIV
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