CN109402108A - The extracting method of RNA in a kind of extracellular vesica - Google Patents
The extracting method of RNA in a kind of extracellular vesica Download PDFInfo
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- CN109402108A CN109402108A CN201710715990.0A CN201710715990A CN109402108A CN 109402108 A CN109402108 A CN 109402108A CN 201710715990 A CN201710715990 A CN 201710715990A CN 109402108 A CN109402108 A CN 109402108A
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- rna
- extracellular vesica
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- 239000003795 chemical substances by application Substances 0.000 claims abstract description 13
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- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- Health & Medical Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the extracting methods of RNA in extracellular vesica a kind of, and the extracting method is the following steps are included: S1: isolating and purifying the extracellular vesica in biological sample, crack the extracellular vesica isolated and purified；S2: organic solvent, which is added, makes RNA enter water phase；S3: alcohol organic solvent is added in the water phase containing RNA that step S2 is obtained；S4: the reagent containing cation is added, then the reagent of the cation is in conjunction with electronegative RNA；S5: the solution containing inertia co-precipitation agent formulations is added；S6: solution obtained in centrifugation step S5 obtains RNA precipitate；S7: washing precipitating, then re-dissolving precipitating is extracellular vesica RNA solution.RNA extraction method of the invention has many advantages, such as that extraction time is short, at low cost, yield is high, with high purity, effect is good, facilitates follow-up test.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to the extracting method of RNA in a kind of extracellular vesica.
Background technique
Extracellular vesica refers to that cell is discharged into the cutter film with phospholipid bilayer encapsulated by structures in external environment
Imitated vesicle structure.Common extracellular vesica includes the types such as excretion body, microcapsule bubble, apoptotic body, the outer film bubble of thallus.
It is that the main reagent that extracts is aided with isothiocyanic acid that the principle of the outer vesica RNA method for extracting of regular growth, which is with phenol, at present
Guanidine or beta -mercaptoethanol, using phenol depolymerizing protein matter and nucleic acid, while denatured protein, since phenol is not enough to inhibit RNA
The activity of enzyme, it is therefore desirable to be denaturalized RNA enzyme using guanidinium isothiocyanate or beta -mercaptoethanol.The reagents such as chloroform are added later and extract sample
RNA in product is centrifuged to obtain the upper strata aqueous phase containing RNA again or chloroform is not added and directly uses in adsorbent material adsorption system
RNA.The combination water on the surface RNA can be replaced using isopropanol etc., wrapping RNA makes its hydrophobic coagulation, and centrifugation is precipitable RNA.It is logical
The aqueous solution washing RNA precipitate for crossing the alcohol such as 70%~75% ethyl alcohol, finally obtains RNA.As shown in Fig. 1, with presently the most
For common commercialization RNA extraction agent trizol, illustrate RNA extractive process.By containing phenol and different sulphur cyanogen in step 1
The trizol lysate sample of sour guanidine makes the RNA in sample is free to be discharged into system；By the way that chloroform and abundant is added in step 2
Concussion makes chloroform extract RNA, obtains the upper strata aqueous phase in step 3 rich in RNA by high speed centrifugation and contains albumen and nucleic acid
Middle layer, lower layer's organic phase；Upper strata aqueous phase is transferred in new pipe by step 4, and the alcohol reagents such as isopropanol are added, and is changed RNA and is existed
Dissolubility in system recycles centrifugation RNA；Since RNA does not dissolve in alcohol, so the ethyl alcohol using 70%~75% is water-soluble
Liquid washs RNA, removes the impurity in RNA precipitate；Supernatant is abandoned in centrifugation, then dissolves RNA with the water for having been removed RNA enzyme.
However, general quantity is all considerably less since extracellular vesica sample is not readily available, then above-mentioned traditional cell is used
There are following disadvantages for outer vesica RNA method for extracting: 1, isopropanol is promoted by capturing the combination water around RNA in step 4
RNA coagulation, since rna content is less in extracellular vesica sample, and RNA is slightly soluble in water, so being only not enough to isopropanol
RNA in precipitation system, this single stepping can bring the RNA of significant proportion to lose, or even cause RNA extracting failure.2, step 6
In since micro RNA precipitate is without the protection of other substances, it is micro in washing process and since RNA is slightly soluble in water
RNA be directly dissolved in water, cause RNA extracting failure.3, RNA precipitate is made by centrifugation in step 6 and step 7, but due to cell
Outer vesica sample is usually seldom, and the RNA amount extracted is also seldom, can not usually see precipitating, is easy during abandoning supernatant
Micro RNA precipitate is siphoned away into discarding and causes RNA extracting failure.
Summary of the invention
In view of this, the object of the present invention is to provide the extracting method of RNA in extracellular vesica a kind of, it is existing to solve
Deficiency in technology.
The purpose of the present invention is be achieved through the following technical solutions:
The extracting method of RNA in a kind of extracellular vesica, the extracting method the following steps are included:
S1: isolating and purifying the extracellular vesica in biological sample, cracks the extracellular vesica isolated and purified；
S2: organic solvent, which is added, makes the RNA in step S1 enter water phase；
S3: alcohol organic solvent being added in the water phase containing RNA that step S2 is obtained, and is mixed；
S4: after the solution in step S3 mixes, it is added immediately the reagent containing cation, is mixed gently, then the cation
Reagent in conjunction with electronegative RNA；
S5: it is added in solution obtained in inertia co-precipitation solution to step S4；
S6: solution obtained in centrifugation step S5 obtains RNA precipitate；
S7: precipitating obtained in washing step S6, then re-dissolving precipitating is extracellular vesica RNA solution.
Further, in step S1, the extracellular vesica includes outside excretion body, microcapsule bubble, apoptotic body and thallus
At least one of film bubble.
Further, in step S1, reagent is extracted using RNA and cracks the extracellular vesica isolated and purified, the RNA is mentioned
Taking reagent includes the reagent containing phenol and/or beta -mercaptoethanol.
Further, in step S2, the organic solvent is chloroform.
Further, in step S3, the alcohol organic solvent includes but are not limited to methanol, ethyl alcohol, propyl alcohol, isopropyl
At least one of alcohol, propylene glycol, glycerine, isoamyl alcohol.
Further, it in step S7, is washed using alcoholic solution, the alcoholic solution includes but are not limited to methanol, second
At least one of alcohol, propyl alcohol, isopropanol, propylene glycol, glycerine, isoamyl alcohol.
Further, in step S4, be added the reagent containing cation final concentration of 0.01M in the solution~
3M。
Further, the described reagent containing cation includes the solution containing monovalent cation and/or contains divalent
The solution of cation.
Further, the reagent containing cation includes but are not limited to sodium acetate, sodium chloride, sodium nitrate, phosphorus
Sour sodium, sodium sulphate, potassium acetate, potassium chloride, potassium nitrate, potassium phosphate, potassium sulfate, magnesium acetate, magnesium chloride, magnesium nitrate, magnesium phosphate, sulphur
At least one of sour magnesium.
Further, the inertia co-precipitation agent formulations in inertia co-precipitation solution include but are not limited to glycogen,
Nucleic acid, ribonucleotide, picodna, deoxyribonucleotide, trehalose, linear polyacrylamide, linear nucleic acid, ringed nucleus
At least one of acid and nucleotide.
Further, in step S6, solution obtained in high speed centrifugation step S5, centrifugal acceleration be 5000g~
15000g, duration 1-60 minute.
Further, it in step S4, after the solution in step S3 mixes, is added immediately at room temperature containing cation
Reagent, mixing gently；
In step S5, after being mixed in step S4, it is added immediately inertia co-precipitation solution at room temperature, mixes gently；?
- 80 DEG C of temperature~stand 1min~16h at room temperature.
Further, the concentration for the inertia co-precipitation solution being added in step S5 is 0.001 μ g/mL~100mg/mL；
The addition volume of the inertia co-precipitation solution and the mass values (μ L/ μ g) of extracellular vesica are as follows: (1: 100)~(1: 1).
Further, in step S1, the biological sample includes but are not limited to cells and supernatant, tissue block, tissue
Liquid, interstitial fluid, tissue leachate, serum, blood plasma, whole blood, intercellular fluid, lymph, cerebrospinal fluid, aqueous humor, hydrothorax, ascites,
Synovia, gastro-intestinal secretion liquid, glandula digestive juice, urine, saliva, sputum, tear, milk, sperm, lung washing liquid, vagina point
At least one in bleeding, vagina washing liquid, fallopian tubal washing liquid, liquor folliculi, amniotic fluid, excrement leachate, thallus culture supernatant etc.
Kind.
The present invention at least has the advantages that
The present invention gives the extracting method of RNA in a kind of extracellular vesica of optimization, have that extraction time is short, cost
It is low, yield is high, with high purity, effect is good, facilitates the advantages that follow-up test.Importantly, the present invention is utilized containing cation
It is negatively charged to neutralize RNA institute in conjunction with RNA for reagent, and enhances combination and package action of the alcohol reagent to RNA, promotes extracellular
The precipitation and precipitating of RNA in vesica sample；The use of inertia settling agent etc. can enhance the sedimentation effect of RNA, use alcohol subsequent
Aqueous solution wash RNA when can also play protection RNA, be reduced or avoided RNA be dissolved into washing solution in, to promote RNA
Yield.Inertia settling agent can also avoid the case where losing micro RNA in operation simultaneously.In short, of the invention
In method can greatly reduce the loss of RNA, significantly improve the extracting concentration and purity of RNA, will not influence subsequent
Therefore test has important application value.
Detailed description of the invention
Fig. 1 is the flow chart that traditional RNA is extracted described in background of invention.
Fig. 2 is the Nanodrop analysis chart of traditional Trizol extracting method acquired results described in the embodiment of the present invention；
Fig. 3 is the Nanodrop analysis chart of extracting method acquired results of the invention described in the embodiment of the present invention.
Specific embodiment
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
It is a part of the embodiment of the present invention, instead of all the embodiments.The detailed description of the embodiment of the present invention presented below is simultaneously
It is not intended to be limiting the range of claimed invention, but is merely representative of selected embodiment of the invention.Based in the present invention
Embodiment, this field commonsense method personnel every other embodiment obtained without creative efforts,
It shall fall within the protection scope of the present invention.
Embodiment 1
The extracting method of RNA in a kind of extracellular vesica, the extracting method the following steps are included:
S1: isolating and purifying the extracellular vesica in biological sample, and the RNA that 1mL is added in the extracellular vesica of 100 μ g takes out
It mentions reagent (such as trizol) and blows and beats the cracking extracellular vesica isolated and purified repeatedly；The extracellular vesica includes excretion body
(exosomes), the outer film bubble (Outer of microcapsule bubble (microvesicles), apoptotic body (apoptoticbody) and thallus
At least one of Membrane Vesicles).
S2: be added 200 μ L chloroforms, acutely concussion mix 15 seconds, be stored at room temperature 5 minutes, under centrifugal acceleration 12000g from
The heart 15 minutes, the RNA in step S1 is made to enter water phase；Upper strata aqueous phase containing RNA is transferred in new centrifuge tube；
S3: the isopropanol isometric with step S2 upper strata aqueous phase is added in the water phase containing RNA that step S2 is obtained,
Gently it is mixed by inversion；
S4: the 100 μ L of reagent containing cation is added in the obtained solution of step S3 at once at room temperature, gently overturns
It mixes, then the reagent of the cation is in conjunction with electronegative RNA, and neutralization RNA institute is negatively charged, and enhancing alcohol organic solvent is (such as different
Propyl alcohol etc.) combination to RNA and package action, promote the precipitation and precipitating of micro RNA in extracellular vesica sample.
The additional amount of reagent containing cation are as follows: the final concentration of 1M or so of the reagent containing cation in the solution；
The reagent for containing cation is magnesium chloride.
S5: the inertia that the concentration for being added immediately 1 μ L at room temperature is 10mg/mL is co-precipitated molten obtained in solution to step S4
It in liquid, is gently mixed by inversion, then stands 30 minutes at room temperature again.Inertia co-precipitation solution in the present invention is that inertia is coprecipitated
Shallow lake reagent, which is dissolved in water or salt ion buffer, to be made.
The inertia co-precipitation reagent is annular nucleic acid and linear polyacrylamide, which, which is co-precipitated reagent, enhances RNA
Sedimentation effect, when the subsequent aqueous solution using alcohol washs RNA, inertia co-precipitation reagent can play protection RNA, reduce or
RNA is avoided to be dissolved into washing solution, to further promote the yield of RNA；And the presence of inertia co-precipitation reagent, it also can be
The case where losing micro RNA is avoided in operating process.
S6: the obtained solution in high speed centrifugation step S5 in the case where centrifugal acceleration is 12000g, centrifugation time 10 minutes,
Just RNA precipitate is obtained；
S7: the ethanol solution that 1mL 75% is added is added to RNA precipitate obtained in step S6, gently overturns, and washs RNA
Precipitating；It is centrifuged 5 minutes in the case where centrifugal acceleration is 8000g again after washing, abandons supernatant, room temperature is hung 10 minutes, reuses 50 μ L
It is extracellular vesica RNA solution that the water for eliminating RNA enzyme, which re-dissolves precipitating,.
In above-mentioned steps S3, isopropanol can be replaced at least one following alcohols: methanol, propyl alcohol, glycerine, propylene glycol,
Isoamyl alcohol etc..In above-mentioned steps S4, the magnesium chloride can be replaced at least one following reagent: sodium chloride, sodium nitrate, phosphoric acid
Sodium, sodium sulphate, sodium acetate, potassium acetate, potassium chloride, potassium nitrate, potassium phosphate, potassium sulfate, magnesium phosphate, magnesium acetate, magnesium nitrate, sulfuric acid
Magnesium etc..In above-mentioned steps S5, the inertia co-precipitation reagent can be used at least one of lower reagent substitution: glycogen, takes off nucleic acid
Oxygen nucleic acid, deoxyribonucleotide, trehalose, linear nucleic acid, ribonucleotide and nucleotide etc..It is described in above-mentioned steps S7
Ethyl alcohol can be used at least one of lower reagent substitution: methanol, propylene glycol, propyl alcohol, isopropanol, glycerine, isoamyl alcohol etc..
Embodiment 2
The extracting method of RNA in a kind of extracellular vesica, the extracting method the following steps are included:
S1: isolating and purifying the extracellular vesica in biological sample, and 1mL RNA is added in the extracellular vesica of 200 μ g and mentions
It takes reagent (such as trizol) to blow and beat repeatedly and cracks the extracellular vesica isolated and purified；The extracellular vesica include excretion body,
At least one of outer film bubble of microcapsule bubble, apoptotic body and thallus；
S2: be added 200 μ L chloroforms, acutely concussion mix 15 seconds, be stored at room temperature 5 minutes, under centrifugal acceleration 12000g from
The heart 15 minutes, the RNA in step S1 is made to enter water phase；Upper strata aqueous phase containing RNA is transferred in new centrifuge tube；
S3: the isopropanol isometric with step S2 upper strata aqueous phase is added in the water phase containing RNA that step S2 is obtained,
Gently it is mixed by inversion；
S4: the 100 μ L of reagent containing cation is added in the obtained solution of step S3 at once at room temperature, gently overturns
It mixes, then the reagent of the cation neutralizes RNA negatively charged, enhancing alcohol organic solvent isopropanols in conjunction with electronegative RNA
Combination and package action to RNA promote the precipitation and precipitating of micro RNA in extracellular vesica sample.
The additional amount of reagent containing cation are as follows: the final concentration of 0.3M of the reagent containing cation in the solution is left
The right side should be sodium acetate containing cationic reagent.
S5: it is added immediately at room temperature molten obtained in the inertia co-precipitation solution to step S4 of the 1 final concentration of 20mg/mL of μ L
It in liquid, is gently mixed by inversion, then stands 30 minutes at -10 DEG C again.
The inertia co-precipitation reagent is glycogen.The inertia is co-precipitated the sedimentation effect of reagent enhancing RNA, in subsequent use
When the aqueous solution of alcohol washs RNA, inertia co-precipitation reagent, which can play protection RNA, RNA is reduced or avoided is dissolved into washing solution
In, to further promote the yield of RNA；And the presence of inertia co-precipitation reagent, can also avoid in operation will be micro
The case where RNA loses.
S6: the obtained solution in high speed centrifugation step S5 in the case where centrifugal acceleration is 12000g, centrifugation time 15 minutes,
Just RNA precipitate is obtained；
S7: the ethanol solution that 1mL 70% is added is added to RNA precipitate obtained in step S6, gently overturns, and washs RNA
Precipitating；It is centrifuged 2 minutes at centrifugal acceleration 12000g again after washing, abandons supernatant, room temperature hangs 15 minutes, reuses 10 μ L and go
It is extracellular vesica RNA solution in addition to the water of RNA enzyme re-dissolves precipitating.
In above-mentioned steps S3, isopropanol can be replaced at least one following alcohols: methanol, ethyl alcohol, propyl alcohol, propylene glycol, third
Triol, isoamyl alcohol etc..In step S4, the sodium acetate can be replaced at least one following reagent: sodium chloride, sodium nitrate, phosphoric acid
Sodium, sodium sulphate, potassium acetate, potassium chloride, potassium nitrate, potassium phosphate, potassium sulfate, magnesium acetate, magnesium chloride, magnesium nitrate, magnesium phosphate, sulfuric acid
Magnesium etc..In step S5, the inertia co-precipitation solution glycogen can be used at least one of lower reagent substitution: nucleic acid, ribose core
Thuja acid, picodna, deoxyribonucleotide, trehalose, linear polyacrylamide, linear nucleic acid, annular nucleic acid and nucleotide
Deng.In step S7, the ethyl alcohol can be used at least one of lower reagent substitution: methanol, propyl alcohol, isopropanol, propylene glycol, the third three
Alcohol, isoamyl alcohol etc..
In Examples 1 and 2, the biological sample includes but are not limited to cells and supernatant, tissue block, tissue fluid, group
Knit liquid, tissue leachate, serum, blood plasma, whole blood, intercellular fluid, lymph, cerebrospinal fluid, aqueous humor, hydrothorax, ascites, synovia,
Gastro-intestinal secretion liquid, glandula digestive juice, urine, saliva, sputum, tear, milk, sperm, lung washing liquid, vaginal secretion,
Vagina washes one's hands and face at least one of liquid, fallopian tubal washing liquid, liquor folliculi, amniotic fluid, excrement leachate, thallus culture supernatant etc..
The concentration and comparison or purity of embodiment 3:RNA
Experimental group: the RNA in extracellular vesica sample is extracted using the method for embodiment 2.
Contrast groups: it is extracted and the identical cell external capsule of experimental group using conventional method (method that background technique is mentioned)
RNA in bubble.
Obtained RNA in Nanodrop test experience group and contrast groups, as shown in table 1 and attached drawing 2~3.Experimental group
The RNA concentration detected is 211.7 ± 23.4ng/ μ L, and that contrast groups is only 47.1 ± 22.6ng/ μ L, and identical type is identical
The outer vesica of the raw cell of content, experimental group are higher by 160ng/ μ L or more than contrast groups, and concentration is higher by 5 times or more, and difference is extremely
Greatly.For the purity (260/280) of obtained RNA, in the art, it is well known that but its purity is at 1.90~2.00
It is best when preferably, close to 2.00, indicate that RNA purity is not good enough or RNA has Partial digestion, this hair when lower than 1.90 or higher than 2.00
The bright improvement by method, improves RNA yield, while being not apparent from influence RNA purity, is shown in Table 1.
The concentration and comparison or purity of 1 RNA of table
| RNA extraction method | Traditional Trizol extracting method | Method in the present invention |
| RNA concentration ng/ μ L | 47.1±22.6 | 211.7±23.4 |
| RNA purity (260/280) | 1.94±0.10 | 1.98±0.05 |
It can be seen that the method for extracting sample RNA compared to traditional trizol from 1 comparing result of table, the present invention is improved
Method can effectively improve the RNA quantity extracted in the outer vesica sample of unit cell, this is extracting cell mainly due to the present invention
Joined when outer vesica RNA containing cation solvent, and RNA have negative electrical charge, cation in solution system with it is micro
RNA is combined, and has neutralized the charge of RNA institute band, so that the alkanol molecules such as isopropanol be made to be easier to combine and wrap up RNA, be RNA with it is molten
Water isolation in liquid system is opened, further polymeric precipitation.Method of the invention simultaneously joined inertia before centrifugation RNA and help
Heavy agent (such as: glycogen), settling agent can effectively assist RNA coagulation, while have micro RNA with visible precipitation form
In centrifugation bottom of the tube, during the washing of subsequent 70%~75% ethanol water, on the one hand inertia settling agent avoids operation
On the other hand the loss of RNA precipitate in the process weakens in the water for even avoiding the washing solution that RNA is dissolved in again.
It should also be noted that, attached drawing 3 at 230nm visible obvious absorption peaks, this is the absorption peak (illustration of settling agent
In be glycogen), and inertia settling agent will not influence RNA subsequent experimental, therefore remove without other steps in follow-up test lazy
Property settling agent.
Embodiment 4: inertia is co-precipitated influence of the reagent to follow-up study
Extracting method and the obtained RNA of contrast groups tradition trizol method that experimental group in embodiment 3 optimizes are used for RT-
qPCR.Reverse transcription is carried out using same amount of RNA, then carries out qPCR amplification experiment.The results are shown in Table 2.
2 Comparison study of table
| RNA extraction method | Trizol | Method in the present invention |
| MiR-16 (Ct value) | 17.36±0.24 | 17.33±0.19 |
| MiR-378-5p (Ct value) | 27.18±0.21 | 27.35±0.42 |
As seen from Table 2, very close using amplification Ct value of the two methods to equivalent cDNA, this shows in the present invention
Co-precipitation reagent of inertia used in RNA method for extracting etc. does not influence follow-up study, that is, will not influence subsequent use.?
It further illustrates, method of the invention has important industrial application value and meaning.
Room temperature in the present invention is the common-sense definition in biological experiment, and about 15~30 ° of temperature typically refers to 24 DEG C
Left and right.In the present invention, the additional amount for being co-precipitated reagent is general are as follows: for extracellular vesica RNA is excessive.
The above description is only a preferred embodiment of the present invention, is not intended to restrict the invention, for those skilled in the art
For, the invention can have various changes and changes.All any modifications made within the spirit and principles of the present invention are equal
Replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. the extracting method of RNA in a kind of extracellular vesica, it is characterised in that: the extracting method the following steps are included:
S1: isolating and purifying the extracellular vesica in biological sample, cracks the extracellular vesica isolated and purified；
S2: organic solvent, which is added, makes the RNA in step S1 enter water phase；
S3: alcohol organic solvent being added in the water phase containing RNA that step S2 is obtained, and is mixed；
S4: after the solution in step S3 mixes, it is added immediately the reagent containing cation, is mixed gently, then the examination of the cation
Agent is in conjunction with electronegative RNA；
S5: it is added in solution obtained in inertia co-precipitation solution to step S4；
S6: solution obtained in centrifugation step S5 obtains RNA precipitate；
S7: precipitating obtained in washing step S6, then re-dissolving precipitating is extracellular vesica RNA solution.
2. the extracting method of RNA in extracellular vesica according to claim 1, it is characterised in that: described in step S1
Extracellular vesica includes at least one of outer film bubble of excretion body, microcapsule bubble, apoptotic body and thallus.In step S1, the life
Object sample include but are not limited to cells and supernatant, tissue block, tissue fluid, interstitial fluid, tissue leachate, serum, blood plasma,
Whole blood, intercellular fluid, lymph, cerebrospinal fluid, aqueous humor, hydrothorax, ascites, synovia, gastro-intestinal secretion liquid, glandula digestive juice, urine
Liquid, saliva, sputum, tear, milk, sperm, lung washing liquid, vaginal secretion, vagina washing liquid, fallopian tubal wash one's hands and face liquid, ovarian follicle
At least one of liquid, amniotic fluid, excrement leachate, thallus culture supernatant etc..
3. the extracting method of RNA in extracellular vesica according to claim 2, it is characterised in that: in step S1, use
RNA extracts reagent and cracks the extracellular vesica isolated and purified, and it includes containing phenol and/or β-sulfydryl that the RNA, which extracts reagent,
The reagent of ethyl alcohol；
In step S2, the organic solvent is chloroform；
In step S3, the alcohol organic solvent includes but are not limited to methanol, ethyl alcohol, propyl alcohol, isopropanol, propylene glycol, the third three
At least one of alcohol, isoamyl alcohol；
It in step S7, is washed using alcoholic solution, the alcoholic solution includes but are not limited to methanol, ethyl alcohol, propyl alcohol, isopropyl
At least one of alcohol, propylene glycol, glycerine, isoamyl alcohol.
4. the extracting method of RNA in extracellular vesica according to claim 3, it is characterised in that: in step S4, institute is added
State the final concentration of 0.01M~3M of the reagent containing cation in the solution.
5. the extracting method of RNA in extracellular vesica according to claim 4, it is characterised in that: it is described containing sun from
The reagent of son includes the reagent containing monovalent cation and/or the reagent containing bivalent cation.
6. the extracting method of RNA in extracellular vesica according to claim 5, it is characterised in that: it is described containing sun from
The reagent of son includes but are not limited to sodium acetate, sodium chloride, sodium nitrate, sodium phosphate, sodium sulphate, potassium acetate, potassium chloride, nitric acid
At least one of potassium, potassium phosphate, potassium sulfate, magnesium acetate, magnesium chloride, magnesium nitrate, magnesium phosphate, magnesium sulfate.
7. the extracting method of RNA in extracellular vesica according to claim 6, it is characterised in that: the inertia co-precipitation
Inertia co-precipitation agent formulations in solution include but are not limited to glycogen, nucleic acid, ribonucleotide, picodna, deoxidation core
Ribotide, trehalose, linear polyacrylamide, linear nucleic acid, annular at least one of nucleic acid and nucleotide.
8. the extracting method of RNA in extracellular vesica according to claim 7, it is characterised in that: in step S6, at a high speed from
Solution obtained in heart step S5, centrifugal acceleration are 5000g~15000g, duration 1-60 minute.
9. the extracting method of RNA in extracellular vesica according to claim 8, it is characterised in that: in step S4, work as step
After solution in S3 mixes, it is added immediately the reagent containing cation, mixing gently at room temperature；
In step S5, after being mixed in step S4, it is added immediately inertia co-precipitation solution at room temperature, mixes gently；In temperature-
80 DEG C~stand 1min~16h at room temperature.
10. the extracting method of RNA in extracellular vesica according to claim 9, it is characterised in that: be added in step S5
Inertia co-precipitation solution concentration be 0.001 μ g/mL~100mg/mL.
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| WO2021216573A1 (en) * | 2020-04-20 | 2021-10-28 | The Regents Of The University Of California | Scalable, easy-to-deploy system and reagents thereof for crispr-associated nuclease-based detection of pathogen genetic material |
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