CN111394296A - Separation method of single exosome and nucleic acid sequencing method - Google Patents
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Abstract
The invention provides a separation method of a single exosome and a nucleic acid sequencing method, belonging to the technical field of biological detection, wherein the separation method comprises the following steps: 1) separating and obtaining a batch of exosomes from the body fluid; 2) mixing the bulk exosomes with an oily liquid drop solution to obtain a suspension; 3) separating the suspension by using a microfluidic channel to obtain a single exosome wrapped by a single droplet; the particle size of a single oily liquid drop in the oily liquid drop solution is 150-250 nm. The method is simple to operate, and can realize the separation of single exosomes and nucleic acid sequencing.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a separation method of a single exosome and a nucleic acid sequencing method.
Background
Exosomes (exosomes) are tiny membrane vesicles with lipid bilayer membranes, the diameter of which is about 30-150 nm, secreted by cells to the extracellular space, and the sizes of exosomes from different sources are different. Exosomes are widely present and distributed in various body fluids, including blood, tears, urine, saliva, milk, ascites, cerebrospinal fluid, and the like; exosomes may also reach other cells or tissues through pathways such as humoral circulation. Exosomes carry and transmit important signal molecules, and participate in intercellular communication by transmitting various signal molecules among cells, thereby regulating various physiological processes. Research has shown that exosomes may be involved in the occurrence and progression of various diseases. The exosome is rich in nucleic acid (microRNA, lncRNA, circRNA, mRNA, tRNA and the like), protein, cholesterol and other substances, and the substances are transported in vivo through the exosome.
The current methods for the isolation of exosomes are ultracentrifugation and ultrafiltration. The exosomes obtained by separation are not high in purity, and batch exosomes are obtained, so that single exosomes cannot be obtained. While exosomes from different sources have certain heterogeneity, and the conventional exosome separation method cannot realize exosome analysis at a single cell level.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for isolating a single exosome and a method for nucleic acid sequencing, which are simple to operate and can realize the isolation of a single exosome and the nucleic acid sequencing.
The invention provides a method for separating a single exosome, which comprises the following steps: 1) separating and obtaining a batch of exosomes from the body fluid; 2) mixing the bulk exosomes with an oily liquid drop solution to obtain a suspension; 3) separating the suspension by using a microfluidic channel to obtain a single exosome wrapped by a single droplet; the particle size of a single oily liquid drop in the oily liquid drop solution is 150-250 nm.
Preferably, the volume ratio of the batch of exosomes to the oily liquid drop solution in the step 2) is 1 (8-12).
Preferably, the volume ratio of the bulk exosomes to the oleaginous droplet solution in step 2) is 1: 10.
Preferably, the mixing temperature in the step 2) is 20-25 ℃.
Preferably, the mixing time in the step 2) is 4-8 min.
Preferably, the microfluidic channel in step 3) is a 300 nm-scale channel.
Preferably, the body fluid comprises one of blood, tears, urine, saliva, milk, ascites, cerebrospinal fluid, nasal secretions and vaginal secretions.
Preferably, the method for separating the bulk exosomes in step 1) comprises one of ultracentrifugation, ultrafiltration, size exclusion chromatography, microfluidics, affinity method and polymer-based co-precipitation method.
The invention provides a nucleic acid sequencing method of a single exosome, which comprises the following steps: and carrying out library construction sequencing or nanopore sequencing on the single exosome obtained by the separation method.
The invention has the beneficial effects that: the invention provides a method for separating a single exosome, which is characterized in that a single oil droplet with a specific particle size is used for wrapping the single exosome, then the single oil droplet is separated by using a microfluidic technology, the separation of the single exosome is realized, the operation is simple, and the separation effect is improved by more than 30%.
Detailed Description
The invention provides a method for separating a single exosome, which comprises the following steps: 1) separating and obtaining a batch of exosomes from the body fluid; 2) mixing the bulk exosomes with an oily liquid drop solution to obtain a suspension; 3) separating the suspension by using a microfluidic channel to obtain a single exosome wrapped by a single droplet; the particle size of a single oily liquid drop in the oily liquid drop solution is 150-250 nm.
In the present invention, a bulk amount of exosomes is isolated from a body fluid. In the present invention, the body fluid preferably includes one of blood, tears, urine, saliva, milk, ascites, cerebrospinal fluid, nasal secretion and vaginal secretion. In the present invention, the method for separating the obtained bulk exosomes preferably comprises one of an ultracentrifugation method, an ultrafiltration method, size exclusion chromatography, an affinity method and a polymer-based co-precipitation method. The present invention is not particularly limited with respect to the specific steps of the ultracentrifugation, ultrafiltration, microfluidics, size exclusion chromatography, affinity, and polymer-based co-precipitation methods, as described in the literature: the research on the method for separating exosomes of Zhangiangxiangccheng wenjiejie Zhangli fruit is advanced, namely, Zhangiaoxing electronic journal, 2018, 04).
According to the invention, the volume ratio of the bulk exosomes to the oily liquid drop solution is 1 (8-12), more preferably 1:10, in the invention, the mixing temperature is preferably 20-25 ℃, the mixing time is preferably 4-8 min, more preferably 5min, in the invention, the mixing is preferably realized by a constant-temperature rotary table shaker, the mixing rotating speed is preferably 10rpm, in the invention, the particle size of a single oily liquid drop in the oily liquid drop solution is preferably 180-220 nm, more preferably 200nm, in the invention, the oily liquid drop is preferably purchased from Invitrogen L ipofectamine3000 under the Seimer flag, and other liposomes meeting the particle size condition of the oily liquid drop can be selected.
After the suspension is obtained, the suspension is separated by a microfluidic channel to obtain a single exosome wrapped by a single droplet. In the invention, the microfluidic channel is preferably a 300 nm-level channel. In the present invention, the microfluidic channel can be selected from 10 Xgenomics, fluidigm C1, or the separation device available from Shanghai medical science and technology (Shanghai) Co., Ltd.
The invention provides a method for sequencing single exosomes by nucleic acid, which comprises the following steps of performing library construction sequencing or nanopore sequencing on the single exosomes obtained by the separation method, wherein the library construction sequencing preferably adopts a SMART-seq & SMART-seq2, CE L-seq, Drop-seq and inDrop sequencing method, and preferably comprises the following steps:
the obtained liquid drop is subjected to cell lysis treatment, primer oligo (dT) is added into lysate for reverse transcription to synthesize cDNA, and several cytosine nucleotides are continuously added at the end to generate a first chain because reverse transcriptase meets a cap structure special for eukaryotic mRNA when reaching the 5' end. Then, a primer containing oligo (dG) is added, annealed, and bonded to the 3' end of the first strand to perform pre-poly (C) overhang hybridization, thereby synthesizing the second strand. The cDNA obtained in the way is subjected to PCR amplification to obtain nanogram-grade DNA, the DNA is sequenced by a nanopore sequencer after being purified, or a library is built by a library-finding kit of companies such as illumina, NEB and the like, and then the sequencing is performed by a next-generation sequencer (refer to http:// www.360doc.com/content/19/0829/13/52645714_857742350. shtml). in the invention, the preferred consignment sequencing company for nanopore sequencing performs the detailed steps as follows: novel nanopore sequencing, product introduction by nanopore corporation. (see https:// nanoporetech. com/cn /)
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Isolation of individual exosomes from blood
Collecting a blood sample, and separating plasma; the exosome in the plasma is separated by the magnetic bead antibody method:
the method comprises the following specific steps:
the blood was centrifuged at 1500rpm for 10min at room temperature, and the supernatant was collected. The supernatant was mixed with 100. mu.l of magnetic beads of CD62 antibody by rotation for 1 hour, and then placed in a magnetic stand for 10 minutes, and the supernatant was discarded and washed 3 times with 500. mu.l of PBS. Mixing the exosome obtained by separation with an oily liquid drop solution at a volume ratio of 1:10 at 25 ℃ for 5min to obtain a suspension;
and (3) placing the suspension in a microfluidic instrument, and separating by using a 300nm channel to obtain a single oily liquid drop wrapping a single exosome.
Example 2
Isolation of individual exosomes from blood
Collecting blood sample, and separating serum; exosomes in serum were isolated by ultracentrifugation:
the method comprises the following specific steps:
centrifuging the liquid at 300g/min for 10min, and collecting supernatant. Centrifuging the supernatant at 2000g/min at normal temperature for 10min, and collecting the supernatant. Centrifuging the supernatant at 10000g/min at normal temperature for 30min, and collecting the supernatant. Centrifuging the supernatant at 200000g/min at room temperature for 70min, and collecting the supernatant. Resuspend with 100. mu.l PBS.
Mixing the exosome obtained by separation with an oily liquid drop solution at a volume ratio of 1:9 at 25 ℃ for 6min to obtain a suspension;
and (3) placing the suspension in a microfluidic instrument, and separating by using a 300nm channel to obtain a single oily liquid drop wrapping a single exosome.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A method of isolation of a single exosome comprising the steps of:
1) separating and obtaining a batch of exosomes from the body fluid;
2) mixing the bulk exosomes with an oily liquid drop solution to obtain a suspension;
3) separating the suspension by using a microfluidic channel to obtain a single exosome wrapped by a single droplet;
the particle size of a single oily liquid drop in the oily liquid drop solution is 150-250 nm.
2. The separation method according to claim 1, wherein the volume ratio of the bulk exosomes to the oily liquid droplet solution in step 2) is 1 (8-12).
3. The separation method according to claim 2, wherein the volume ratio of the bulk exosomes to the oleaginous liquid droplet solution in step 2) is 1: 10.
4. The separation method according to claim 1 or 2, wherein the temperature of the mixing in step 2) is 20 to 25 ℃.
5. The separation method according to claim 3, wherein the mixing time in the step 2) is 4-8 min.
6. The separation method according to claim 1, wherein the microfluidic channel in step 3) is a 300nm channel.
7. The separation method of claim 1, wherein the body fluid comprises one of blood, tears, urine, saliva, milk, ascites, cerebrospinal fluid, nasal secretions, and vaginal secretions.
8. The separation method according to claim 1, wherein the method for separating the obtained bulk exosomes in step 1) comprises one of ultracentrifugation, ultrafiltration, size exclusion chromatography, microfluidics, affinity, and polymer-based co-precipitation.
9. A method of nucleic acid sequencing of a single exosome, comprising the steps of: performing library-building sequencing or nanopore sequencing on a single exosome isolated by the isolation method of any one of claims 1-8.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160370265A1 (en) * | 2013-12-03 | 2016-12-22 | bioMérieux | Method for isolating exosomes |
| US20170065978A1 (en) * | 2014-03-14 | 2017-03-09 | University Of Kansas | Non-invasive monitoring cancer using integrated microfluidic profiling of circulating microvesicles |
| CN110462064A (en) * | 2017-04-18 | 2019-11-15 | 深圳华大生命科学研究院 | The method and its application of microorganism detection are carried out based on excretion body nucleic acid |
| CN110777048A (en) * | 2019-11-01 | 2020-02-11 | 上海市第六人民医院 | High-flux single exosome separation device |
-
2020
- 2020-04-01 CN CN202010251176.XA patent/CN111394296A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160370265A1 (en) * | 2013-12-03 | 2016-12-22 | bioMérieux | Method for isolating exosomes |
| US20170065978A1 (en) * | 2014-03-14 | 2017-03-09 | University Of Kansas | Non-invasive monitoring cancer using integrated microfluidic profiling of circulating microvesicles |
| CN110462064A (en) * | 2017-04-18 | 2019-11-15 | 深圳华大生命科学研究院 | The method and its application of microorganism detection are carried out based on excretion body nucleic acid |
| CN110777048A (en) * | 2019-11-01 | 2020-02-11 | 上海市第六人民医院 | High-flux single exosome separation device |
Non-Patent Citations (1)
| Title |
|---|
| 刘娜 等: "基于微流控技术的外泌体分离方法的研究进展", 《生物技术通报》 * |
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Application publication date: 20200710 |