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Method and kit used for determining human circulation blood exosome circular RNA

An exosome and circular technology, which is applied in the field of a method for measuring exosome circular RNA in human circulating blood and its kit, can solve the problem of not being able to accurately quantify exosome circular RNA in human circulating blood, etc. question

Pending Publication Date: 2019-07-02
中国人民解放军海军军医大学国家肝癌科学中心
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Problems solved by technology

However, there is currently no method that can accurately quantify exosomal circular RNA in human circulating blood.

Method used

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  • Method and kit used for determining human circulation blood exosome circular RNA
  • Method and kit used for determining human circulation blood exosome circular RNA
  • Method and kit used for determining human circulation blood exosome circular RNA

Examples

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Embodiment 1

[0042] This embodiment is a method for isolating and quantifying exosomal circular RNA from human plasma samples, which includes the following steps:

[0043] 1. Blood sample collection and processing

[0044] 1. Use an EDTA-containing anticoagulant tube (Gongdong, Cat. No. GD050EK2) to collect 10 mL of venous blood from patients or normal people.

[0045] Centrifuge at 2.4°C, 3000rpm for 10min, collect the supernatant, and repeat the centrifugation once. The collected supernatant is the plasma. Be careful not to absorb the middle and lower layers of cells and solid components. The volume of plasma collected from 10 mL of venous blood was about 5 mL, and stored at -80°C.

[0046] 2. Human plasma exosome isolation and RNA extraction

[0047] For exosome isolation and RNA extraction in human plasma, use the exoRNeasy Serum / Plasm Midi kit (QIAGEN, product number: 77044), and refer to the reagent manufacturer’s method to improve the method. The detailed process is as follows:

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Abstract

The invention relates to a method and a kit used for determining human circulation blood exosome circular RNA. The method includes the following steps: collecting human venous blood, performing centrifugation, and collecting a blood plasma sample; performing exosome separation and total RNA extraction on the blood plasma sample to obtain a total RNA sample; determining the concentration of the RNAsample, and taking an appropriate amount of the RNA sample for performing a reverse transcription reaction to obtaining a reverse transcription product cDNA; and performing PCR amplification on the reverse transcription product cDNA by using an amplification primer shown as SEQ ID NO:1-SEQ ID NO:2, and analyzing and determining the content of circular RNA in exosome according to an amplificationproduct. The invention also relates to an application of human serum exosome circular RNA Circ-CDYL as a diagnostic marker for liver cancer. The steps of human plasma exosome separation and RNA extraction are optimized, a specific primer is designed for target gene, the method can separate exosome from a small amount of human plasma samples, rapidly quantifies expression quantity of the circular RNA, and is used for diagnosis of the liver cancer and monitoring of high-risk populations by analyzing a difference of the circular RNA between patients with tumors and healthy people.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a method for measuring exosome circular RNA in human circulating blood and a kit thereof. Background technique [0002] Circular RNA (circular RNA, circRNA) is a new member of the RNA family that is different from traditional linear RNA. It has a closed circular structure. Biologically functional non-coding RNAs. [0003] Exosomes are lipid bilayer membranous vesicles, which are released into the extracellular space or biological fluid after the fusion of multivesicular body (MVB) and cell membrane in living cells, with a size ranging from 30 to Between 100nm, it contains various components such as protein, RNA, microRNA and DNA fragments. Studies have clearly shown that exosomes contain a large number of circular RNAs. In addition to cell-derived exosomes, studies have also found that human circulating blood exosomes also contain a large number of co...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6886C12Q1/6851C12Q2600/158C12Q2600/178C12Q2600/166
Inventor 王红阳李亮危晏平梁驰任一彬
Owner 中国人民解放军海军军医大学国家肝癌科学中心
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