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
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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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