CN114369595A - Purified separating liquid of cerebral cortex microvessel and great vessel RNA and separating method thereof - Google Patents
Purified separating liquid of cerebral cortex microvessel and great vessel RNA and separating method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The application belongs to the technical field of biology, and particularly relates to a cerebral cortex microvessel and great vessel RNA purification separation solution and a separation method thereof. The application provides a purified separating medium of cerebral cortex microvessels and macrovascular RNA, which comprises: a first buffer, a second buffer and a third buffer; wherein the first buffer solution comprises 4-hydroxyethyl piperazine ethanesulfonic acid HEPES, an RNase inhibitor and Hank's balanced salt solution HBSS; the second buffer solution comprises glucan and the first buffer solution, and the molecular weight of the glucan is 65000-75000; the third buffer comprises animal serum and the first buffer. The application provides a purifying separation solution of cerebral cortex microvascular and macrovascular RNA and a separation method thereof, which can effectively solve the technical problems of low purity of cerebral cortex blood vessels, low purity of extracted RNA and low integrity in the prior art.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to a cerebral cortex microvessel and great vessel RNA purification separation solution and a separation method thereof.
Background
With the rapid development of molecular biology, the research on nucleic acid is increasing. Cerebral cortical vascular RNA is widely used in medical research, particularly in clinical diagnostics. Extracting high-quality total RNA from cerebral cortex blood vessel samples is the basis of technologies such as downstream sequencing, qPCR, chip detection and the like. However, there are technical difficulties in extracting high-quality total RNA from cerebral cortical blood vessels, and the difficulty in storing RNA is a major bottleneck in the research of gene expression sets. RNA is very fragile and difficult to store, and due to the extensive existence of RNases in nature, including RNases in tissues and pathogens, RNA starts to degrade within a few minutes at room temperature, and is completely degraded into small fragments which cannot be detected within 30 minutes, so that the experimental research of RNA is limited.
The current method for purifying animal cerebral cortex blood vessel RNA is mainly to homogenize the brain tissue, filter and separate the micro-vessel or the macro-vessel tissue by screens with different apertures, and then extract the tissue RNA. However, the purity of the purified vascular tissue in the prior art is generally not high, and the conventional method lacks measures for protecting RNA, so that the obtained RNA has incomplete structure and poor quality, and subsequent RNA sequencing cannot be performed.
Disclosure of Invention
In view of this, the present application provides a separation liquid for purifying RNA in microvasculature and macrovascular of cerebral cortex and a separation method thereof, which can effectively solve the technical problems of low purity of cerebral cortex blood vessels purified in the prior art, low purity of RNA extracted therefrom and low integrity.
In a first aspect, the present application provides a purified and isolated solution of RNA in microvasculature and macrovascular in cerebral cortex, comprising:
a first buffer, a second buffer and a third buffer;
wherein the first buffer solution comprises 4-hydroxyethyl piperazine ethanesulfonic acid HEPES, an RNase inhibitor and Hank's balanced salt solution HBSS;
the second buffer solution comprises glucan and the first buffer solution, and the molecular weight of the glucan is 65000-75000;
the third buffer comprises animal serum and the first buffer.
Specifically, the dextran in the second buffer solution is used to control the solution density, and when dextran with a molecular weight of more than 75000 is used, since the solution density is high, the vascular tissue precipitated by centrifugation is less, the separation efficiency is low, and the centrifugation time is long; when dextran with a molecular weight less than 65000 is used, vascular tissue and other tissue components such as myelin sheaths cannot be separated. Therefore, the dextran with the molecular weight of 65000-75000 is used, so that the tissue structures of the isolated cerebral cortex microvessels and great vessels are complete, the structure of the purified RNA molecule is complete, and the RNA sequencing can be carried out.
Specifically, the molecular weight of the glucan is 70000.
Specifically, the blood vessels can be classified into capillary vessels (less than 10 μm), microvessels (10-50 μm), and macrovessels (more than 50 μm) according to their diameters. The cerebral cortex is rich in various types of blood vessels, and the separation and purification of the microvasculature and the macrovascular are realized by homogenizing cerebral tissues and then sequentially passing through a filter membrane with a preset aperture to obtain the microvasculature and the macrovascular of the cerebral cortex, for example, the microvasculature and the macrovascular of the cerebral cortex can sequentially pass through filter membranes with apertures of 100 mu m and 20 mu m, and filter residues obtained by washing are blood vessels (the microvasculature and the macrovascular of the cerebral cortex respectively) with corresponding diameters.
In another embodiment, in the first buffer solution, the Hank's balanced salt solution HBSS is a solvent, the HEPES and the rnase inhibitor are solutes, and the final concentration of the 4-hydroxyethylpiperazine ethanesulfonic acid HEPES is 1% to 5%; the final concentration of the RNase inhibitor is 0.1-0.5%.
In another embodiment, in the first buffer, the rnase inhibitor is selected from one or more of diethyl pyrocarbonate, DEPC, guanidinium isothiocyanate, and a vanadyl ribonucleoside complex.
In another embodiment, in the second buffer solution, the first buffer solution is a solvent, the glucan is a solute, and the mass percentage of the glucan is 150 g/L-200 g/L.
Specifically, the mass percentage of the glucan is 180 g/L.
In another embodiment, in the third buffer solution, the first buffer solution is a solvent, and the animal serum is used in an amount of 1 v/v% to 10 v/v%.
In another embodiment, in the third buffer, the animal serum is selected from one or more of goat serum and albumin thereof, donkey serum and albumin thereof, and bovine serum and albumin thereof.
Specifically, the first buffer solution, the second buffer solution and the third buffer solution in the purified separation solution of the present application are all added with rnase inhibitors, which can inhibit rnases from air in the solution and remove rnases in each extraction step to prevent RNA degradation.
Specifically, HEPES is used as tissue antioxidant, prevents partial protein degradation, and adjusts pH; HBSS is used as a main solvent for dissolving glucan and maintaining the activity of cerebral cortex microvascular and macrovascular tissues; animal serum is used as a substance for maintaining the activity of cerebral cortex microvascular and macrovascular tissues.
In a second aspect, the present application provides a method for isolating RNA from microvasculature and macrovascular of the cerebral cortex, comprising:
step 2, mixing the cortical layer homogenate sediment with a second buffer solution, then centrifuging, and removing the jelly and the supernatant of the upper layer to obtain cerebral cortex vascular sediment;
step 3, mixing the cerebral cortex vascular precipitate with a third buffer solution, then filtering the mixed solution through a filter membrane, washing and collecting cerebral cortex microvessels passing through the filter membrane and cerebral cortex macrovessels attached to the filter membrane by using the third buffer solution;
wherein the first buffer solution is the first buffer solution of the purified separation solution; the second buffer solution is the second buffer solution of the purified separation solution; the third buffer solution is the third buffer solution of the purification separation solution.
Specifically, in the step 3, the filtration is performed twice, the first filtration is performed by a filter membrane with a pore size of 100 μm, and the second filtration is performed by a filter membrane with a pore size of 20 μm; the first filtration is used to filter large vessels and the second filtration is used to filter micro-vessels.
Specifically, step 3 specifically includes:
1. 100 μm, 20 μm filters were placed on the filter holder on top of the beaker, respectively, and equilibrated with ice-cold third buffer.
2. The mixture was passed through a 100 μm filter and the 100 μm filter was washed with ice-cold third buffer while collecting the cortical vascular filtrate on the filter.
3. The 100 μm filter was withdrawn using forceps and immersed in a beaker containing a third buffer. The sediment obtained is cerebral cortex large blood vessel tissue by gentle shaking and uniform dispersion.
4. The cerebral cortical vascular filtrate collected in step 2 was passed through a 20 μm filter and the 20 μm filter was washed with ice-cold third buffer.
5. The 20 μm filter was withdrawn using forceps and immersed in a beaker containing a third buffer. Gently shaken and evenly dispersed, and the obtained precipitate is cerebral cortex microvascular tissue.
6. And respectively transferring the obtained cerebral cortex macrovascular tissues and cerebral cortex microvascular tissues into a centrifuge tube, centrifuging for 5 minutes at 2000g, removing supernate, and concentrating and purifying vascular tissues to obtain the cerebral cortex macrovascular tissues and the cerebral cortex microvascular tissues.
In particular, the cortical layer of the Brain can be obtained according to techniques disclosed in the prior art, such as Purification of mouse Brain Vessels, J Vis exp.2015Nov 10; (105) e53208.doi: 10.3791/53208.
Specifically, the method for obtaining the cortical layer of the brain comprises the following steps:
1. mice were deeply anesthetized with 2% isoflurane.
2. Intracardiac perfusion was performed with 20ml pbs to eliminate blood content.
3. The skin was incised from neck to nose with a scalpel and pulled open. All hairs were then removed by rinsing with PBS.
4. Opening the skull: first scissors are inserted in front of the olfactory bulb and then the skull is divided into two parts by opening the scissors.
5. The brain was carefully removed with a spoons and forceps.
6. The brains were transferred to ice-cold first buffer.
7. Removing pia mater from brain, and separating cortex to obtain cortex of brain.
In another embodiment, in the step 1, the temperature of the centrifugation is 0-4 ℃, the centrifugal acceleration of the centrifugation is 2000-5000 g, and the time of the centrifugation is 10-20 minutes.
Specifically, in the step 1, the temperature of the centrifugation is 4 ℃, the centrifugal acceleration of the centrifugation is 2000g, and the time of the centrifugation is 10 minutes.
In another embodiment, in the step 2, the temperature of the centrifugation is 0-4 ℃, the centrifugal acceleration of the centrifugation is 3000-5000 g, and the time of the centrifugation is 15-30 minutes.
Specifically, in step 2, the temperature of the centrifugation is 4 ℃, the centrifugation acceleration of the centrifugation is 4400g, and the centrifugation time is 15 minutes.
In another embodiment, in step 3, the pore size of the filter membrane is 20 μm to 100 μm.
The application uses glucan with specific molecular weight to obtain cerebral cortex microvessels and great vessels with high purity and complete structures, and combines a buffer solution with specific components to extract total RNA with high purity and high integrity from the cerebral cortex microvessels and the great vessels.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the immunofluorescence results of the purified cerebral cortex microvessels as provided in example 1 of the present application;
FIG. 2 shows the immunofluorescence results of the purified cerebral cortex microvessels as provided in example 1 of the present application;
FIG. 3 shows the immunofluorescence results of the purified cerebral cortex microvessels provided in example 1 of the present application;
FIG. 4 shows the immunofluorescence results of the purified cerebral cortex microvessels provided in example 1 of the present application;
FIG. 5 shows the immunofluorescence results of the purified cerebral cortex microvessels provided in example 1 of the present application;
FIG. 6 shows the immunofluorescence results of the purified cerebral cortex microvessels provided in example 1 of the present application;
FIG. 7 shows the data of cellular markers of RNA of cerebral cortex microvasculature after purification, as provided in example 1 of the present application.
Detailed Description
The application provides a purifying separation liquid of cerebral cortex microvascular and macrovascular RNA and a separation method thereof, which are used for solving the technical defects of low purity of purified cerebral cortex vessels, low purity of extracted RNA and low integrity in the prior art.
The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The raw materials and reagents used in the following examples are commercially available or self-made.
HBSS used in the following examples was purchased from ThermoFisher Scientific, cat # 14175095; HEPES purchased from ThermoFisher Scientific under item number 15630080; DEPC is purchased from Biyunyan, and the cargo number is ST 036; dextran purchased from Sigma-Aldrich, cat # 31390; BSA was purchased from Biyun, and the cat # was ST 023.
Preparation method of first buffer solution B1: to 150mL of HBSS were added 1.5mL of 1M HEPES and 150. mu.L of DEPC. After placement, it was kept on ice and covered with a sealing film to avoid air contamination.
Preparation method of second buffer solution B2: 20mL of the first buffer was added with 3.6g of dextran (molecular weight 70000). After placement, it was kept on ice and covered with a sealing film to avoid air contamination.
Preparation method of third buffer B3: 100mL of the first buffer was added to 1g of BSA. After placement, it was kept on ice and covered with a sealing film to avoid air contamination.
Example 1
The embodiment of the application provides a method for separating cerebral cortex microvessels from macrovascular RNA, which comprises the following steps:
1. mice were deeply anesthetized with 2% isoflurane.
2. Intracardiac perfusion with 20ml PBS to eliminate blood content
3. The skin was incised from neck to nose with a scalpel and pulled open. All hairs were then removed by rinsing with PBS.
4. Opening the skull: first scissors are inserted in front of the olfactory bulb and then the skull is divided into two parts by opening the scissors.
5. The brain was carefully removed with a spoons and forceps.
6. The brains were transferred to ice-cold beakers of B1 solution.
7. The brain was then separated into the cortical layers after removal of the pia, and the cortical layers of the brain were placed in a fresh beaker of cold B1 solution.
8. The cortical layer of the brain was minced and homogenized until no apparent tissue mass was visible.
9. The homogenate was transferred to a 50ml plastic centrifuge tube and centrifuged at 2000g for 10 minutes at 4 ℃.
10. The supernatant was discarded to give a cortical homogenate precipitate. To the cortical homogenate pellet, 20mL of ice-cold B2 solution was added and shaken vigorously for 1 min.
11. A second centrifugation at 4400g was carried out at 4 ℃ for 15 minutes.
12. Holding the tube and slowly rotating, carefully separating the upper layer of colloidal matter from the tube wall, and allowing the remaining supernatant to flow out along the tube wall to obtain cerebral cortex vascular precipitate.
13. The inner wall of the tube is wiped by absorbent paper to remove all residual liquid, so as to avoid contact with the bottom of the tube for precipitation.
14. Adding 1mL of ice-cold B3 solution into the cerebral cortex vascular sediment, blowing and beating the sediment up and down by using a low adsorption suction head until the sediment is uniformly dispersed, then adding 5mL of B3 solution, further uniformly mixing and dispersing to obtain a dispersion liquid containing the sediment. This step operates keeping the centrifuge tubes on ice.
15. 100 μm, 20 μm filters were placed on the filter holder at the top of the beaker, respectively, and equilibrated with 10mL of ice-cold B3 solution.
16. The dispersion containing the precipitate from step 14 was poured onto a 100 μm filter and the filter was rinsed with 20mL of ice-cold B3 solution while collecting the cortical vascular filtrate on the filter.
17. The filter was withdrawn using clean forceps and immediately immersed in a beaker containing the B3 solution. The sediment obtained is cerebral cortex large blood vessel tissue by gentle shaking and uniform dispersion.
18. The cerebral cortical vascular filtrate collected in step 16 was poured onto a 20 μm filter and the filter was rinsed with 20mL of ice-cold B3 solution.
19. The filter was withdrawn using clean forceps and immediately immersed in a beaker containing the B3 solution. Gently shaken and evenly dispersed, and the obtained precipitate is cerebral cortex microvascular tissue.
20. Respectively transferring the purified cerebral cortex macrovascular tissues and cerebral cortex microvascular tissues into a plastic centrifuge tube, centrifuging for 5 minutes at 2000g, pouring out supernatant, concentrating the purified vascular tissues to obtain the cerebral cortex macrovascular tissues and the cerebral cortex microvascular tissues, and carrying out next step of RNA extraction and morphological verification.
Immunofluorescence validation of the purified cerebral cortex microvasculature obtained above was carried out, and the results of the markers of vascular endothelial cells, CD31(Red), and cell nucleus (Blue), are shown in fig. 1 to 6, and the Red signal in fig. 1 to 6 is the marker of vascular endothelial cells, CD 34.
Extracting RNA of the purified cerebral cortex microvasculature obtained above, performing reverse transcription, and performing purity identification by using a fluorescence quantitative PCR method, wherein the results are shown in figure 7 for Purifield vessels, and figure 7 for Unputrified, which is obtained by using the existing documents of Purification of mouse BrainVessels, JVIS Exp.2015Nov 10; (105) e53208.doi 10.3791/53208, and Purifield vessels in FIG. 7 are the cellular marker data of the purified RNA of cerebral cortex microvessels obtained in this example. As can be seen from FIG. 7, the method of the present application successfully isolated and purified RNA from cerebral cortex microvessels and macrovessels.
To sum up, the technical defects that the purity of cerebral cortex microvessels and great vessels obtained in the prior art is not high, and high-purity and high-integrity total RNA in the cerebral cortex microvessels and the great vessels cannot be obtained are solved. The application uses glucan with specific molecular weight to obtain cerebral cortex microvessels and great vessels with high purity, and adopts specific buffer solution to extract total RNA with high purity and high integrity from the cerebral cortex microvessels and the great vessels.
The foregoing is only a preferred embodiment of the present application and it should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.
Claims (10)
1. A purified separating medium for RNA of cerebral cortex microvessels and macrovascular is characterized by comprising:
a first buffer, a second buffer and a third buffer;
wherein the first buffer solution comprises 4-hydroxyethyl piperazine ethanesulfonic acid HEPES, an RNase inhibitor and Hank's balanced salt solution HBSS;
the second buffer solution comprises glucan and the first buffer solution, and the molecular weight of the glucan is 65000-75000;
the third buffer comprises animal serum and the first buffer.
2. The purification separation solution according to claim 1, wherein the Hank's balanced salt solution HBSS is a solvent and the final concentration of HEPES 4-hydroxyethylpiperazine ethanesulfonic acid in the first buffer solution is 1% to 5%; the final concentration of the RNase inhibitor is 0.1-0.5%.
3. The purification isolate of claim 1, wherein in the first buffer, the rnase inhibitor is selected from one or more of diethyl pyrophosphate DEPC, guanidine isothiocyanate, and a vanadyl riboside complex.
4. The purification and separation solution of claim 1, wherein the first buffer solution is a solvent and the dextran is present in an amount of 150 to 200g/L by mass.
5. The purification and separation solution of claim 1, wherein the first buffer solution is a solvent and the animal serum is used in an amount of 1 v/v% to 10 v/v% in the third buffer solution.
6. The purification separator according to claim 1, wherein in the third buffer, the animal serum is selected from one or more of goat serum and albumin thereof, donkey serum and albumin thereof, and bovine serum and albumin thereof.
7. A method for separating RNA from microvasculature and macrovascular of cerebral cortex, which is characterized by comprising the following steps:
step 1, putting the cortex of the brain into a first buffer solution, shearing and homogenizing the cortex, then centrifuging, and removing supernatant to obtain cortex homogenate sediment;
step 2, mixing the cortical layer homogenate sediment with a second buffer solution, then centrifuging, and removing the jelly and the supernatant of the upper layer to obtain cerebral cortex vascular sediment;
step 3, mixing the cerebral cortex vascular precipitate with a third buffer solution, then filtering the mixed solution through a filter membrane, washing and collecting cerebral cortex microvessels passing through the filter membrane and cerebral cortex macrovessels attached to the filter membrane by using the third buffer solution;
wherein the first buffer solution is the first buffer solution of the purified separation solution according to any one of claims 1 to 6; the second buffer solution is the second buffer solution of the purified separation solution of any one of claims 1 to 6; the third buffer solution is the third buffer solution for the purified separation solution according to any one of claims 1 to 6.
8. The separation method according to claim 7, wherein in the step 1, the temperature of the centrifugation is 0 to 4 ℃, the centrifugal acceleration of the centrifugation is 2000 to 5000g, and the time of the centrifugation is 10 to 20 minutes.
9. The separation method according to claim 7, wherein in the step 2, the temperature of the centrifugation is 0 to 4 ℃, the centrifugal acceleration of the centrifugation is 3000 to 5000g, and the time of the centrifugation is 15 to 30 minutes.
10. The separation method according to claim 7, wherein in step 3, the pore size of the filter membrane is 20 to 100 μm.
Priority Applications (1)
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