Method for accurately obtaining exosome miRNA (micro Ribonucleic Acid)

An exosome and precise technology, applied in the field of RNA detection, can solve the problems affecting the detection sensitivity and accuracy, the accuracy and purity of miRNA, etc., to achieve comprehensive and accurate expression profiles, avoid bias, and achieve the effect of accurate counting

Inactive Publication Date: 2017-05-10
朱小兰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In view of this, the purpose of the present invention is to provide a method for accurate acquisition of exosomal miRNA, so as to solve the problem that the accuracy and purity of miRNA obtained in the prior art are not enough, which affects the sensitivity and accuracy of later detection

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  • Method for accurately obtaining exosome miRNA (micro Ribonucleic Acid)

Examples

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Embodiment 1

[0031] Draw 10ml of peripheral blood from the subject into an EDTA anticoagulant tube, gently invert it up and down 7 times and mix thoroughly, and perform the following treatment within 5 hours on the day of blood collection: Use the Wako exosome extraction kit to obtain exosomes from human peripheral blood.

[0032] After all the exosomes were isolated, the total RNA in the exosomes was extracted. Specifically, the Tiangen RNAprep pure blood total RNA extraction kit was used. After the total RNA was extracted, the total RNA was dissolved back into 14 μL of RNase-freewater, and then Preliminary quantification was performed based on Qubit2.0 (Invitrogen, QubitRNAHSAssayKits).

[0033] Connect the specific unique label linker to the exosome total RNA to obtain the total RNA with the specific linker; the specific unique label linker is:

[0034] 5′-rAppCTGTAGGCACCATCAATATTGCGCACGA-NH 2 3′

[0035] Mix the total RNA with specific adapters and other reverse transcription materia...

Embodiment 2

[0037] Put 10ml of human body fluid into an EDTA anticoagulant tube, invert up and down 6 times and mix thoroughly, and perform the following treatment within 4 hours on the day of blood collection: Use Wako exosome extraction kit to obtain exosomes.

[0038] After all the exosomes were isolated, the total RNA in the exosomes was extracted. Specifically, a total RNA extraction kit was used. After the total RNA was extracted, the total RNA was dissolved back into 10 μL of RNase-freewater, and then based on Qubit2.0 ( Invitrogen, QubitRNAHSAssayKits) for preliminary quantification.

[0039] Connect the specific unique label linker to the exosome total RNA to obtain the total RNA with the specific linker; the specific unique label linker is:

[0040] 5′-rAppCTGTAGGCACCATCAATGAACTCGTCGA-NH 2 3′

[0041] Mix the total RNA with specific adapters and other reverse transcription materials, mix the solution evenly, place it on a PCR machine, and incubate at 43°C for 55 minutes to rev...

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Abstract

The invention provides a method for accurately obtaining an exosome miRNA (micro Ribonucleic Acid). The method comprises the following steps: 1) separating the exosome from a sample to be separated; 2) extracting RNA from the exosome, so as to obtain exosome total RNA; 3) connecting a specific unique label connector onto the exosome total RNA, so as to obtain the total RNA with a specific connector; 4) carrying out reverse transcription by taking the total RNA with the specific connector as a template to synthesize cDNA (complementary Deoxyribonucleic Acid), so as to obtain a reverse transcription product; 5) recycling a cDNA fragment of 150bp to 170bp in the reverse transcription product through TBE-PAGE (Tetrabromoethane-Polyacrylamide Gel Electrophoresis) to obtain the miRNA. The method provided by the invention not only avoids preference between molecules in an amplification process, but also effectively realizes accurate counting of the single template; miRNA molecules lower than 10 copies can be accurately detected.

Description

technical field [0001] The invention relates to the field of RNA detection, in particular to a method for accurately obtaining exosome miRNA. Background technique [0002] Exosomes were first discovered in sheep reticulocytes in 1983, and Johnstone named them "exosomes" in 1987. Exosomes are disc-shaped vesicles with a diameter of 40-100 nm, which can be secreted by various cells under normal and pathological conditions. Exosomes are mainly derived from the multivesicular bodies formed by the invagination of intracellular lysosomal particles, and are released into the extracellular matrix after the fusion of the multivesicular outer membrane and the cell membrane. [0003] When exosomes were discovered, they were once considered a way for cells to excrete waste. With the deepening of research on exosomes, including their biological origin, distribution, material composition and transportation, and intercellular signal transduction, Exosomes have been found to have diverse ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 朱小兰许文林李月峰陈小芳殷新明徐宇浩魏虹
Owner 朱小兰
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