Method for detecting nucleic acid molecules in exosomes

A detection method and technology of nucleic acid molecules, applied in the field of detection of RNA and DNA contained in exosomes, can solve problems such as unfavorable extraction and quantification of RNA or DNA, loss of RNA or DNA molecules, influence analysis of results, etc., so as to overcome mutual inhibition. , Improve detection sensitivity and save consumption

Pending Publication Date: 2021-07-23
GUANGZHOU FULENGEN
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the commonly used extraction of exosomal RNA or DNA is mostly based on phenol extraction or chromatographic column-based methods. Such methods will not only cause the loss of some RNA or DNA molecules, directly affect the subsequent analysis of results, and this method is relatively complicated, time consuming
In particular, the amount of various exosome samples is generally small, which is not conducive to the extraction and quantification of RNA or DNA

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  • Method for detecting nucleic acid molecules in exosomes
  • Method for detecting nucleic acid molecules in exosomes
  • Method for detecting nucleic acid molecules in exosomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Simple and rapid detection of exosomal RNA in Hek293 cell culture medium by one-step method

[0068] In this example, the recovery efficiency of exosome sample RNA was mainly compared using different methods. The exosome direct lysis (one-step) method of the present invention (ie, A, as the experimental group) was compared with the conventional RNA extraction method (multi-step) method (ie, B, as the control group). For the convenience of comparison, in the experimental group and the control group, except that the exosome RNA sample extraction steps are different (respectively direct lysis method and conventional RNA extraction method), other operating steps are the same, for example, both use the method of the present invention RT-For-All reverse transcription reaction for exosome nucleic acid detection. The specific operation is as follows:

[0069] 1. Experimental materials

[0070] (1) Main equipment: centrifuge (Thermo), PCR instrument (Takara), real-...

Embodiment 2

[0101] Example 2: One-step method for simple and rapid detection of exosomal RNA in mesenchymal stem cell (MSC) culture medium

[0102] In this example, the method of the present invention is completely applied to the detection of exosomes of mesenchymal stem cells (MSC). According to the latest literature reports, the marker miRNAs and housekeeping RNAs in the exosomes of mesenchymal stem cells (MSCs) were selected for detection, including Mir23a-3p, Mir125b, Mir1246a, etc.

[0103] In this example, in the reverse transcription step of the exosome RNA detection method, the RT-For-All reverse transcription reaction (ie, A, experimental group) in the method of the present invention was combined with the conventional miRNA reverse transcription reaction (ie, B, Control 1) was compared with a conventional mRNA reverse transcription reaction (ie, C, Control 2).

[0104] Among them, the conventional mRNA reverse transcription reaction (i.e., control group 2) can only detect some m...

Embodiment 3

[0124] The operation method of Example 3 is similar to that of the experimental group in Example 1. The sample is ExoCT exosome lysate obtained by lysing exosomes with ExoCt lysis buffer. The difference is that reverse transcriptase and PolyA polymerase in the reverse transcription reaction system The concentrations of , miRNA Adapter and random primers are different, as shown in Table 1. In the reverse transcription reaction system, the concentration of reverse transcriptase is 1U / μL reaction system, and the concentration of the PolyA polymerase is 0.05U / μL reaction system; the concentration of miRNA Adapter in the RT buffer solution in the reverse transcription reaction is 0.2 μM, and the concentration of random primers is 0.2 μM. Test results image 3 As shown, it is found that it can achieve similar technical effects.

[0125] Table 1 embodiment 3-6 reverse transcription reaction system component concentration

[0126] RT-For-ALL Example 3 Example 4 Exam...

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Abstract

The invention relates to a method for simply, conveniently and quickly detecting nucleic acid molecules in exosomes. The method comprises the following steps: cracking the exosomes in a sample to release the nucleic acid molecules of the exosomes; and performing reverse transcription reaction: after the exosomes are cracked, the nucleic acid molecules are not extracted, or after the nucleic acid molecules are extracted, reverse transcription reaction of all RNA is carried out, namely RT-For-All, so that all RNA in the exosomes is simultaneously subjected to reverse transcription in the same reaction; and a reverse transcription buffer comprising a universal adapter (e.g., miRNA Adapter) and a random primer is used in the reverse transcription reaction. The invention also provides a kit for detecting the nucleic acid molecules in the exosomes. Contained components are released by directly dissolving the exosomes, RNA and DNA do not need to be extracted, and reverse transcription of all RNA is directly carried out in the same reverse transcription reaction. Various RNA and DNA can be detected from trace exosomes.

Description

technical field [0001] The invention relates to a simple and rapid method for detecting the characteristics of exosomes, and mainly relates to the detection of RNA and DNA contained in the exosomes. Background technique [0002] Exosomes are extracellular vesicles with a size of about 30-150 nm and a lipid bilayer membrane structure. Exosomes were discovered in sheep reticulocytes as early as 1983, but have since been thought of as a way for cells to excrete waste. With the in-depth study of exosomes in recent years, it has been found that they carry a large amount of important information such as proteins, lipids, DNA and RNA (miRNA, mRNA, lncRNA, circRNA), not only participate in intercellular communication and information exchange, according to exosomes It also plays an important role in many aspects such as antigen presentation, tumor growth and migration, and tissue damage repair. [0003] Almost all cells in the human body can secrete exosomes under normal or patholo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2521/107C12Q2521/101C12Q2525/191C12Q2521/537C12Q2527/125C12Q2527/143
Inventor 徐学明冯韵田金金冯菲菲
Owner GUANGZHOU FULENGEN
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