Application and preparation kit of a kind of exosome microRNA

An exosome and kit technology, applied in the field of application and preparation of kits, can solve the problems of small amount of research samples, inability to comprehensively and objectively reflect the situation of circulating microRNA, lack of multi-center verification, etc., achieving good stability and saving time. Labor costs, easy-to-obtain results

Active Publication Date: 2022-07-22
THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the current research on circulating microRNA as a detection indicator still has the following deficiencies: (1) Most of the studies only selected microRNAs with dysregulated expression in skin lesions reported by previous studies as candidate indicators, which may not be able to fully and objectively reflect the situation of circulating microRNAs; (2) Some studies have small sample size and lack of multi-center verification

Method used

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  • Application and preparation kit of a kind of exosome microRNA
  • Application and preparation kit of a kind of exosome microRNA
  • Application and preparation kit of a kind of exosome microRNA

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1: Detection of microRNA expression in serum / urine exosome samples of systemic scleroderma patients and healthy control serum / urine exosome samples

[0038] In this example, 7 cases of systemic scleroderma patient serum, 15 cases of morning urine samples, 4 cases of healthy control serum, and 16 cases of morning urine samples were used.

[0039] 1. Processing of serum and urine samples:

[0040] Serum 2ml / morning urine 50ml, processed within 4h, centrifuged at 3000rpm / h at room temperature for 15min, and the supernatant was separated.

[0041] 2. Extraction of exosomes:

[0042] All exosome extractions were performed according to ExoQuick-TC TM Procedure provided by Exosome Precipitation Solution (SBI).

[0043] The collected exosome pellet was resuspended in 10ul of enzyme-free water and stored at -80°C.

[0044] 3. Extract total RNA from exosomes:

[0045] Add 1000ul TRIzol LS Reagent (Ambion) to 10ul exosome resuspended liquid, and mix by pipetting up an...

Embodiment 2

[0076] Example 2: Detection kit of the present invention

[0077] Including urine exosome extraction system, exosome total RNA extraction system, reverse transcription system, amplification system and relative quantitative internal reference standardization system; the kit is to quantitatively detect urine exosome microRNA molecular markers microRNA-8064 to determine whether there is systemic scleroderma.

[0078] Specifically include the following:

[0079] The urine exosome extraction system includes ExoQuick-TC;

[0080] The exosome total RNA extraction system includes TRIzolLS Reagent, chloroform, isopropanol, 75% alcohol, RNase-free water (enzyme-free water);

[0081] The reverse transcription system includes RTase mix, 5×RTase Buffer, Uni-RT Primer;

[0082] The amplification system includes Forward Primer (10 μM), Uni-Reverse Primer (10 μM), 2×SYBR Green Mix, RNase-free water.

[0083] The relative quantitative internal reference standardization system consists of c...

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Abstract

The invention relates to the field of biotechnology, and discloses an application and preparation kit of exosome microRNA. The present invention proves the abnormal expression of exosome microRNA-8064 in systemic scleroderma samples through biochemical tests, suggesting that it can be a biomarker for systemic scleroderma detection, and can be applied to the preparation of related products , disease diagnosis; exosomal microRNA‑8064 diagnosis of SSc can more timely reflect the disease state of patients with systemic scleroderma, avoid complicated detection in the past, save time and labor costs, and facilitate clinicians to take personalized prevention and treatment plans in a timely manner; Exosome samples are easy to obtain, more clinically maneuverable and less invasive, and exosomal microRNAs are more stable and easier to detect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for the application and preparation of exosome microRNA. Background technique [0002] Systemic sclerosis (SSc) is an autoimmune disease involving multiple systems, mainly manifesting as fibrosis, sclerosis and atrophy of connective tissue of skin and internal organs. The prevalence of this disease is 0.24‰, which is second only to rheumatoid arthritis and lupus erythematosus among connective tissue diseases in my country. The median survival time is about 11 years, and it is even lower if multiple internal organs are involved. In addition to serious threats to life, the quality of life of patients is also significantly reduced. The specific pathogenesis of SSc is still unclear, and the existing related pathogenesis theories include the immune theory, the abnormal collagen synthesis theory, and the vascular theory. Currently, T lymphocytes, B lymphocytes, fibroblasts, vascula...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6851
CPCC12Q1/6883C12Q1/6851C12Q2600/158C12Q2600/178C12Q2531/113C12Q2525/207C12Q2545/101C12Q2563/107
Inventor 肖嵘喻江帆曾茁桐陆前进赵明吴海竞邓前程史雅倩唐冰思邹璞玉唐瑞曾光明杨洋汪文军汤湘
Owner THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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