CN109679947A - RNA purification kit and the method for being enriched with miRNA - Google Patents
RNA purification kit and the method for being enriched with miRNA Download PDFInfo
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- CN109679947A CN109679947A CN201910020384.6A CN201910020384A CN109679947A CN 109679947 A CN109679947 A CN 109679947A CN 201910020384 A CN201910020384 A CN 201910020384A CN 109679947 A CN109679947 A CN 109679947A
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Abstract
This application involves a kind of RNA purification kits, including acid processing glass powder and aaerosol solution, which, which handles glass powder, has 325 mesh or thinner granularity, and the aaerosol solution is using ultrapure water as solute, the buffer of chaotropic salt and 0.05-0.2M comprising 0.2-1M, pH 5-7.The application further relates to a kind of method that miRNA is enriched with from the aqueous solution containing total serum IgE, the aqueous solution containing total serum IgE is handled including using the acid in RNA purification kit of the present invention to handle the RNA purified reagent that glass powder and aaerosol solution are made into, is enriched with miRNA from total serum IgE through subsequent processing.The RNA purification kit and miRNA enrichment method of the application is at low cost, relatively easy, miRNA DNA purity height is operated, extraction loss is small, the reagent and data waste that subsequent high pass amount is built in the sequencing procedure of library are reduced, blood plasma or the extraction purification and selective enrichment of serum miRNA are particularly suitable for.
Description
Technical field
The present invention relates to biochemical purification technical fields, and in particular to a kind of RNA purification kit and a kind of enrichment miRNA's
Method.
Background technique
MiRNA (MicroRNA) is the single-stranded tiny RNA of a kind of non-coding, is generally made of 18-23 nucleotide.As miniature
Molecule is adjusted, miRNA is inhibited protein translation or directly led by the 3 '-UTR region specific bonds with mRNA (mRNA)
MRNA degradation is caused, in the expression of post-transcriptional level regulation protein.It is all kinds of heavy that miRNA participates in proliferation, differentiation, apoptosis of cell etc.
Big biochemical process, performance in human body are exactly the occurrence and development process in some major diseases such as cardiovascular disease, tumour
Middle performance adjusting function.The study found that mature miRNA can be stable in the presence of in blood plasma or serum, it can be cardiovascular disease
The diagnosis and prognosis of disease etc. provide important clue.
In order to detect the expression of miRNA in blood plasma or serum, it is necessary first to be extracted from blood plasma or serum sample
To purity is high, the miRNA of total amount foot.Blood plasma or serum RNA extraction method common at present is broadly divided into two classes, and one kind is special
Extracts kit, such as QIAampCircuLating NucleicAcidKit of QIAGEN, Thermo
mirVanaTMPARISTMKit etc., another kind of is conventional total RNA extraction reagent, as Thermo TrizolLS,
TrizolReagent etc..
The side that the QIAampCircuLatingNucleicAcidKit of QIAGEN mainly uses silicate-base plasma membrane to adsorb
Formula is enriched with the RNA in blood plasma or serum.Blood plasma or serum sample are after lysate is handled, by being centrifuged nucleic acid
Specificity is integrated on QIAamp centrifugal column, and by subsequent cleaning step, by sample protein, PCR mortifier,
The removal such as pollutant obtains the nucleic acid samples of purifying finally by elution.Meanwhile it can be with the matched RNase- of binding reagents box
It is digested on the column of FreeDNaseSet progress DNA, it is ensured that without DNA pollution in the nucleic acid samples of acquisition.
The mirVana of ThermoTMPARISTMThe method that Kit separating kit uses glass fibre absorption, can be from group
It knits, separate various types RNA in cell and blood plasma and serum, such as total serum IgE, mRNA, miRNA, small molecules interference RNA (siRNA)
With microRNA in core (snRNA) etc..This kit uses organic solvent extractive technique, and using special binding soln with
It washs solution and carries out glass fiber filter purifying, to obtain the RNA sample of high-purity.
The TrizolLS and TrizolReagent of Thermo belongs to conventional RNA and extracts reagent, and main component phenol is group
It knits or cell cracking agent, also containing RNA enzyme (RNase) inhibitor such as 8-hydroxyquinoline, guanidinium isothiocyanate, beta -mercaptoethanols.
In Trizol extraction process, the RNA aqueous solution of high-purity, and the process for passing through chloroform are obtained using acid aqueous environment
The impurity such as DNA, protein are removed, ethyl alcohol or isopropanol alcohol precipitation are finally used, obtain total serum IgE sample.
In current existing technology, the kit extracted dedicated for blood plasma or serum RNA is expensive, although can obtain
The higher blood plasma of purity or serum RNA product, but often complex steps, yield is lower, and without RNA type or fragment length
Specificity.In addition, based on filter membrane adsorption filtration enrichment method, be often limited to the manufacture craft of film, cause fragment length compared with
Loss of the small RNA in centrifugal process, is unfavorable for the detection of miRNA.In subsequent detection process, no length specificity
The other types RNA for extracting non-miRNA in product often occupies more reagent and data resource, leads to the further of cost
It increases.Conventional RNA extracts Trizol LS and the Trizol Reagent of reagent such as Thermo, and price is inexpensively, general to obtain
Rate is also higher, but poor for the removing effect of pollutant, while also only can not distinguish different type, no by extracting process
With the RNA of fragment length.
Summary of the invention
It is an object of the invention to overcome the defect of the above-mentioned prior art, a kind of improved RNA purification kit is provided,
It can be used for being enriched with miRNA from the aqueous solution containing total serum IgE.
Therefore, in a first aspect, the present invention provides a kind of RNA purification kit, which includes acid processing
Glass powder and aaerosol solution, which, which handles glass powder, has 325 mesh or thinner granularity, which is molten with ultrapure water
Matter, the buffer of chaotropic salt and 0.05-0.2M comprising 0.2-1M, pH5-7.
In specific embodiments of the present invention, which handles glass powder and is prepared by the method with following steps:
(I) glass material is ground into the glass powder with 325 mesh or thinner granularity；
(II) by the glass powder with 1g:(3-5) w/v of ml is added in ultrapure water, stirs and evenly mixs, stand 90-
180 minutes, resulting supernatant was centrifuged 5-15 minutes at 5000-8000rpm after then standing, and abandoned supernatant；
(III) resulting glass powder being precipitated with 1g:(3-5) w/v of ml is resuspended in the acid solution of 20-40%
In, it stands overnight, resulting acid blend is centrifuged 5-15 minutes at 5000-8000rpm after then standing, and abandons supernatant；
(IV) by the precipitating of resulting glass powder again with 1g:(3-5) w/v of ml is resuspended in ultrapure water, stands
90-180 minutes, resulting supernatant was centrifuged 5-15 minutes at 5000-8000rpm after then standing, and abandoned supernatant；
(V) it repeats step (IV) repeatedly, until abandoning supernatant after pH value is in neutrality, it is heavy to obtain resulting glass powder to the end
It forms sediment, i.e., the acid handles glass powder.
In specific embodiments of the present invention, which can be nitric acid solution, hydrochloric acid solution or sulfuric acid solution.
In specific embodiments of the present invention, the chaotropic salt be lithium chloride, potassium rhodanide, sodium perchlorate or they
Combination.
In specific embodiments of the present invention, which is ammonium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, acetic acid
Sodium or their combination.
In specific embodiments of the present invention, which also includes the EDTA of 0.05-2mM.
In further preferred embodiment of the present invention, the aaerosol solution include the lithium chloride of 0.5M, 0.1M ammonium chloride and
The EDTA of 1mM.
In some embodiments of the present invention, acid processing glass powder and the aaerosol solution are divided in different reagent bottles
In.Before use, acid processing glass powder and the aaerosol solution are mixed now to match RNA purified reagent, wherein the acid handles glass
The w/v of glass powder and the aaerosol solution is 1g:(3-5) ml.
In other embodiments of the invention, acid processing glass powder is blended in same in advance with the aaerosol solution
RNA purified reagent is made into reagent bottle, it is 1g:(3-5 which, which handles glass powder and the w/v of the aaerosol solution) ml.
In second aspect, the present invention provides a kind of method that miRNA is enriched with from the aqueous solution containing total serum IgE, this method
Include:
(1) aqueous solution containing total serum IgE is provided；
(2) acid in the RNA purification kit of first aspect present invention is handled into glass powder and aaerosol solution is mixed with existing
With RNA purified reagent, wherein the w/v of acid processing glass powder and the aaerosol solution is 1g:(3-5) ml；Or it provides
The RNA purified reagent prepared in the RNA purification kit of first aspect present invention；
(3) contain the RNA purified reagent that 0.6-1 times of volume is added in the aqueous solution of total serum IgE to this, mix, obtain the
One mixed liquor is placed 5-15 minutes at 4 DEG C；
(4) first mixed liquor is centrifuged 5-15 minutes at 4 DEG C, 5000-8000rpm, takes supernatant, 0.75-1 is added
The isopropanol of times volume, and 10-20 μ g glycogen is added, obtain the second mixed liquor；
(5) second mixed liquor is placed at least 2h at -80 DEG C, then melted, at 4 DEG C, 10000-15000rpm from
The heart 30-40 minutes, discard supernatant liquid；
(6) 70% ethyl alcohol of pre-cooling is added to resulting sediment, mixes, is centrifuged at 4 DEG C, 10000-15000rpm
10-15 minutes, discard supernatant liquid；
(7) step (6) are repeated once, discards supernatant liquid, have the centrifuge tube of sediment in 4000-6000rpm bottom
After lower brief centrifugation 5s, liquid is thoroughly discarded supernatant using small-range pipettor, drying at room temperature 2-5 minutes, obtains being enriched miRNA
Sediment；
(8) optionally, suitable pyrocarbonic acid diethyl ester (DEPC) processing water is added to the sediment for being enriched miRNA,
After being stored at room temperature 2-5 minutes, mixes, obtain the aqueous solution for being enriched miRNA.
In specific embodiments of the present invention, the step of aqueous solution of the offer containing total serum IgE, includes:
(A) sample containing RNA is provided；
(B) the Trizol Reagent of 2-2.5 parts by volume is added to the sample for containing RNA of 1 parts by volume, mixes, room temperature
Stand 5-10 minutes；
(C) isopropanol of 0.8-1 parts by volume is added into step (B) resulting mixed liquor, mixes, is stored at room temperature 5-10 points
Clock；
(D) step (C) resulting mixed liquor is centrifuged 10-15 minutes at 4 DEG C, 10000-15000rpm, takes supernatant
Into new centrifuge tube；
(E) chloroform of 2-2.5 parts by volume is added to the gained supernatant of 1 parts by volume, mixes, is stored at room temperature 5-10 minutes；
(F) step (E) resulting mixed liquor is centrifuged 10-15 minutes at 4 DEG C, 10000-15000rpm, takes supernatant
Into new centrifuge tube；
(G) it repeats step (E) and (F) once, obtains the aqueous solution for containing total serum IgE.
In a preferred embodiment of the invention, the sample containing RNA is blood plasma or serum.
Beneficial effects of the present invention:
The key of RNA purification kit of the invention is glass powder therein under the acidic environment of Trizol to sheet
The suction-operated of section nucleic acid.Of the invention from the method for being enriched with miRNA in the aqueous solution containing total serum IgE, the sample containing RNA
It is cracked by Trizol Reagent, large fragment DNA, the RNA for obtaining the aqueous solution cracking release containing total serum IgE are inhaled by glass powder
Attached removal obtains high-purity, the miRNA enriched product of high segment concentration degree after subsequent processing.In addition, the present invention is containing total
In the preparation of the aqueous solution of RNA, is extracted in process in common Trizol, increase the premix of the isopropanol of certain volume, make
During chloroform protein and DNA etc. precipitated and separated effect it is more preferable.
RNA purification kit of the invention and the method that miRNA is enriched with from the aqueous solution containing total serum IgE can significantly drop
Low reagent cost, operation is relatively easy, extracts sample miRNA at high speed for big flux and provides a simple and effective solution party
Case；The ratio for extracting miRNA in product can be significantly improved, subsequent high pass amount is reduced and builds reagent and data in the sequencing procedure of library
Waste, further saves testing cost；MiRNA DNA purity is high, and extraction process loss is small, and it is less to can be adapted for sample size
The extraction of precious sample.The RNA purification kit and this method are particularly suitable for extraction purification and the choosing of blood plasma or serum miRNA
The enrichment of selecting property.
Detailed description of the invention
Fig. 1 shows blood plasma miRNA method for extraction and purification according to an embodiment of the present invention and QIAamp CircuLating
Nucleic Acid Kit and mirVanaTMPARISTMThe comparison of Kit method for extraction and purification, wherein from left to right, the first band
Build library as a result, the single concentration of library band for the embodiment of the present invention, brightness is high, second and third strip be all Marker, beQuick-Load pBR322DNA-MspI Digest in the reagent preparation box of Small RNA multisample library
Standard items, Article 4 band are that QIAamp CircuLating Nucleic Acid Kit extracts the library of product building as a result, the
Five bands are mirVanaTMPARISTMKit extracts the result in product building library；
Fig. 2 shows the sequence of the resulting blood plasma miRNA product of blood plasma miRNA method for extraction and purification according to an embodiment of the present invention
Column distribution of lengths.
Specific embodiment
Below by specific embodiment and in conjunction with attached drawing, invention is further described in detail.
The present invention provides a kind of RNA purification kit, which includes acid processing glass powder and suspend molten
Liquid, which, which handles glass powder, has 325 mesh or thinner granularity, which includes 0.2-1M's using ultrapure water as solute
The buffer of chaotropic salt and 0.05-0.2M, pH 5-7.
In the present invention, term " acid processing glass powder " refers to silicate glass material as main component (such as quartz
Sand) ground obtained glass powder material, the glass powder of acid treated acquisition.It can be public by glass treatment technical field
The technology and equipment (such as miniature planetary ball mill) known grinds glass material, then with the well known sieve of field of powder preparation
Divide technology and equipment to sieve to obtain granularity as 325 mesh or thinner glass powder material.Then, with the nitric acid of 20-40%, salt
Acid or the sulfuric acid treatment glass powder material obtain purer silicate glass powder to remove alkali composition therein.
The acid, which handles glass powder, has suction-operated to large fragment nucleic acid under the acidic environment of Trizol.Of the invention
From the method for being enriched with miRNA in the aqueous solution containing total serum IgE, the aqueous solution containing total serum IgE is split by Trizol Reagent
It solving, the large fragment DNA, RNA in the RNA of release handle glass powder Adsorption by the acid in RNA purification kit of the invention,
To obtain the miRNA enriched product of high-purity, high segment concentration degree.
In the present invention, term " ultrapure water " refers to that resistivity reaches the water of 18M Ω * cm (25 DEG C), is almost to go deoxygenation
What impurity typically without RNA enzyme, organic, nothing in light water can be prevented almost without with the water of all atoms other than hydrogen
The influence of machine and colloidal impurity to experimental result.The preparation of ultrapure water is well known to water-treatment technology field.
In the present invention, term " aaerosol solution " refer to for suspend acid processing glass powder solution, with formed for from
The RNA purified reagent of miRNA is enriched in aqueous solution containing total serum IgE.In some embodiment party of RNA purification kit of the invention
In case, acid processing glass powder and aaerosol solution are divided in different reagent bottles.Before use, by acid processing glass powder and it is somebody's turn to do
Aaerosol solution mixing is now to match RNA purified reagent, and wherein the w/v of acid processing glass powder and the aaerosol solution is 1g:
(3-5)ml.In other embodiments of RNA purification kit of the invention, which handles glass powder and the aaerosol solution
It is blended in the same reagent bottle in advance and is made into RNA purified reagent, which handles the bulking value of glass powder and the aaerosol solution
Than for 1g:(3-5) ml.Require it that acid processing glass powder can be made outstanding for a long time it should be pointed out that term " aaerosol solution " does not represent
Float on wherein.The RNA purified reagent that acid processing glass powder and aaerosol solution mix, may during long-time is stood
There is number acid to handle glass powder Precipitation, but this has no effect on the use of the RNA purified reagent, is as long as mixing when in use
It can.
In the present invention, term " chaotropic salt " includes lithium chloride, potassium rhodanide, sodium perchlorate or their combination.From
Salt solution provides monovalent cation, reduces Si-substrate surface and nucleic acid molecules electronegativity to reduce electrostatic repulsion, while reducing silicon
Matrix and nucleic acid molecules hydration levels (being exactly chaotropic), so that it is real to allow the siloxy of nucleic acid molecules and Si-substrate surface to form hydrogen bond
Now adsorb.Lithium chloride, potassium rhodanide, sodium perchlorate can achieve the same or similar chaotropic effect in the present invention.Chaotropic salt
Concentration in the aaerosol solution is 0.2-1M, such as 0.2M, 0.3M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M.
In the present invention, term " buffer " include ammonium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium acetate or it
Combination.Buffer provides and maintains the pH environment of acid processing glass powder absorption large fragment RNA.Ammonium chloride, disodium hydrogen phosphate,
Sodium dihydrogen phosphate, sodium acetate can achieve the same or similar buffering effect in the present invention.Buffer is in the aaerosol solution
Concentration be 0.05-0.2M, such as 0.05M, 0.08M, 0.1M, 0.15M, 0.2M.
In the present invention, aaerosol solution maintains the slant acidity environment of pH 5-7, for example, pH 5.2, pH 5.5, pH 6.0,
pH 5.5,pH 6.8.Phosphate group in the siloxy and nucleic acid molecules of silicon matrix belongs to weak acid to a certain extent, acid
Environment can inhibit weak acid to hydrolyze, and reduce surface negative charge, while hydration levels can also reduce.
In the present invention, which also may include the EDTA of 0.05-2mM, for example, 0.1mM, 0.5mM, mM, 1mM,
The EDTA of 1.5mM, 2mM.EDTA is that may be present (despite the presence of possibility pole by chelating RNA purified reagent as chelating agent
Its is low) magnesium ion or other ions, to inhibit there may be the RNA enzyme of (extremely low despite the presence of possibility), with prevent from
After being enriched with miRNA in aqueous solution containing total serum IgE, miRNA is potentially decomposed.
The present invention also provides the acid processing glass powder preparation method, the preparation method the following steps are included:
(I) glass material is ground into the glass powder with 325 mesh or thinner granularity；
(II) by the glass powder with 1g:(3-5) w/v of ml is added in ultrapure water, stirs and evenly mixs, stand 90-
180 minutes, resulting supernatant was centrifuged 5-15 minutes at 5000-8000rpm after then standing, and abandoned supernatant；
(III) resulting glass powder being precipitated with 1g:(3-5) w/v of ml is resuspended in the acid solution of 20-40%
In, it stands overnight, resulting acid blend is centrifuged 5-15 minutes at 5000-8000rpm after then standing, and abandons supernatant；
(IV) by the precipitating of resulting glass powder again with 1g:(3-5) w/v of ml is resuspended in ultrapure water, stands
90-180 minutes, resulting supernatant was centrifuged 5-15 minutes at 5000-8000rpm after then standing, and abandoned supernatant；
(V) it repeats step (IV) repeatedly, until abandoning supernatant after pH value is in neutrality, it is heavy to obtain resulting glass powder to the end
It forms sediment, i.e., the acid handles glass powder.
In step (I), (such as the miniature planetary ball of technology and equipment well known to glass treatment technical field can be passed through
Grinding machine) glass material is ground, then material sieving technology and equipment well known to field of powder preparation are come to sieve to obtain granularity be 325
Mesh or thinner glass powder.
In step (II), glass powder forms the suspension of glass powder after stirring and evenly mixing with ultrapure water, and the effect of standing is
Remove the excessive glass powder of particle.
In step (III), acid solution is to obtain purer glassy silicate for washing the alkali composition taken out in glass powder
Glass powder.It can be using nitric acid, hydrochloric acid or the sulfuric acid of 20-40% as the acid solution.
The method that the present invention also provides a kind of to be enriched with miRNA from the aqueous solution containing total serum IgE, this method comprises:
(1) aqueous solution containing total serum IgE is provided；
(2) acid in the RNA purification kit of first aspect present invention is handled into glass powder and aaerosol solution is mixed with existing
With RNA purified reagent, wherein the w/v of acid processing glass powder and the aaerosol solution is 1g:(3-5) ml；Or it provides
The RNA purified reagent prepared in the RNA purification kit of first aspect present invention；
(3) contain the RNA purified reagent that 0.6-1 times of volume is added in the aqueous solution of total serum IgE to this, mix, obtain the
One mixed liquor is placed 5-15 minutes at 4 DEG C；
(4) first mixed liquor is centrifuged 5-15 minutes at 4 DEG C, 5000-8000rpm, takes supernatant, 0.75-1 is added
The isopropanol of times volume, and 10-20 μ g glycogen is added, obtain the second mixed liquor；
(5) second mixed liquor is placed at least 2h at -80 DEG C, then melted, at 4 DEG C, 10000-15000rpm from
The heart 30-40 minutes, discard supernatant liquid；
(6) 70% ethyl alcohol of pre-cooling is added to resulting sediment, mixes, is centrifuged at 4 DEG C, 10000-15000rpm
10-15 minutes, discard supernatant liquid；
(7) step (6) are repeated once, discards supernatant liquid, have the centrifuge tube of sediment in 4000-6000rpm bottom
After lower brief centrifugation 5s, liquid is thoroughly discarded supernatant using small-range pipettor, drying at room temperature 2-5 minutes, obtains being enriched miRNA
Sediment；
(8) optionally, suitable pyrocarbonic acid diethyl ester (DEPC) processing water is added to the sediment for being enriched miRNA,
After being stored at room temperature 2-5 minutes, mixes, obtain the aqueous solution for being enriched miRNA.
In the present invention, term " aqueous solution containing total serum IgE " refers to the preprocessed obtained water phase of the sample containing RNA
Solution can make miRNA therein from total and the RNA purified reagent of RNA purification kit of the invention is added thereto
Enrichment comes out in RNA, achievees the purpose that extraction purification contains the miRNA in the sample of RNA.
In step (4), nucleic acid molecules are that the state of hydration dissolves in water phase, and addition isopropanol can seize and core
The hydrone that acid molecule combines, so that nucleic acid is precipitated.Glycogen can assist nucleic acid to precipitate to be formed, while in subsequent ethanol cleaning
Play the role of tracer in the process.
In step (5), -80 DEG C of favors low temperature is in the coagulation of nucleic acid molecules, while chilling process solution state occurs
Change, it is easier to precipitating be precipitated.
In step (6), 70% ethyl alcohol assists in removing the organic and inorganic impurity in nucleic acid extractive.
In step (7), tentatively discarded supernatant after liquid still due to during ethyl alcohol cleans, some liquid
Rest on centrifugation tube wall, and impurity can be contained in these liquid, therefore in order to thoroughly discard residual supernatant, using instantaneously from
Residual liquid on tube wall is all collected into tube bottom by the method for the heart, is reused small-range pipettor and is sucked.
In step (8), it may not deposited in water using the RNA that DEPC processing water can protect as much as possible extraction to obtain
RNA enzyme degradation.DEPC processing water is prepared by the following method in advance: by DEPC (a kind of strong RNase inhibitor)
1000 times are diluted in ultrapure water, are stirred overnight on magnetic stirring apparatus, are then decomposed using high-pressure sterilizing pot high temperature and pressure
DEPC finally obtains the ultrapure water of no RNA enzyme.
The present invention also provides a kind of methods for preparing the aqueous solution containing total serum IgE, method includes the following steps:
(A) sample containing RNA is provided；
(B) the Trizol Reagent of 2-2.5 parts by volume is added to the sample for containing RNA of 1 parts by volume, mixes, room temperature
Stand 5-10 minutes；
(C) isopropanol of 0.8-1 parts by volume is added into step (B) resulting mixed liquor, mixes, is stored at room temperature 5-10 points
Clock；
(D) step (C) resulting mixed liquor is centrifuged 10-15 minutes at 4 DEG C, 10000-15000rpm, takes supernatant
Into new centrifuge tube；
(E) chloroform of 2-2.5 parts by volume is added to the gained supernatant of 1 parts by volume, mixes, is stored at room temperature 5-10 minutes；
(F) step (E) resulting mixed liquor is centrifuged 10-15 minutes at 4 DEG C, 10000-15000rpm, takes supernatant
Into new centrifuge tube；
(G) it repeats step (E) and (F) once, obtains the aqueous solution for containing total serum IgE.
In the present invention, term " sample containing RNA " refers to material wherein comprising RNA nucleic acid substances, can be packet
The biomaterial of the nucleic acid substances containing RNA is also possible to the non-biological material that pickup has RNA nucleic acid substances.Include RNA nucleic acid substances
Biomaterial for example have cytoplasm, nucleus, cell, tissue, excrement, body fluid (such as sweat, blood, urine, sperm) etc..
Preferably, the biomaterial of RNA nucleic acid substances is processed blood, such as blood plasma and serum.That is, in the present invention, containing
There is the sample of RNA to be preferably blood plasma and serum.The method for preparing blood plasma and serum from blood is well known to those skilled in the art.
In step (A), the sample containing RNA can be obtained by the sampling technique of this field routine.
In step (B), Trizol Reagent for cracking the sample containing RNA, release large fragment RNA, DNA and
miRNA。
In step (C), the premix of isopropanol can make in step (E) protein and DNA etc. during chloroform
Precipitated and separated effect it is more preferable.
The invention will be further described by the following examples, these embodiments are intended for for example, not
It is intended to limit the invention in any way.
The preparation of embodiment 1:RNA purification kit
I. the preparation (subpackage type) of acid processing glass powder
Industrial quartz sand is bought, is ground into 325 mesh or thinner glass powder with miniature planetary ball mill (Focucy).It takes
50g glass powder is resuspended in the ultrapure water of 200ml, stands 90min after stirring and evenly mixing at room temperature.Supernatant is transferred to 50ml
In centrifuge tube, 6000rpm is centrifuged 10min, to remove the excessive glass powder of particle.After outwelling supernatant, by resulting glass powder weight
It is suspended from 25% nitric acid of 15ml, stands overnight at room temperature, to remove alkaline impurities present in industrial quartz sand.It will mixing
6000rpm is centrifuged 10min to liquid at room temperature, carefully sucks supernatant.Resulting glass powder is resuspended using 20ml ultrapure water to precipitate,
After standing 20min at room temperature, 6000rpm is centrifuged 10min, abandons supernatant.It repeats last action 4-6 times, until after pH value is in neutrality,
Supernatant is abandoned in centrifugation, retains glass powder precipitating to get acid processing glass powder is arrived.
II. the preparation of aaerosol solution
2.115g lithium chloride and 0.535g ammonium chloride are weighed, is dissolved in 100mL ultrapure water, adjusting pH value to pH 5.5,
The aaerosol solution comprising the lithium chloride of about 0.5M and the ammonium chloride of about 0.1M is made.
The assembling of III.RNA purification kit
Acid processing glass powder made above and aaerosol solution are respectively charged into individual reagent bottle, said together with kit
Bright book is packaged into RNA purification kit together.When in use, by 1g:(3-5) w/v of ml, from two reagent bottles point
Acid processing glass powder and aaerosol solution are not taken, are mixed, are obtained RNA purified reagent.
The preparation (subpackage type) of embodiment 2:RNA purification kit
The present embodiment is substantially the same manner as Example 1, and difference is the preparations of aaerosol solution: weigh 2.115g lithium chloride,
0.535g ammonium chloride and 0.037g EDETATE SODIUM, are dissolved in 100mL ultrapure water, adjust pH value to pH 6.5, are made comprising about
The aaerosol solution of the EDTA of the ammonium chloride of the lithium chloride of 0.5M, about 0.1M and about 1mM.
The preparation (mixed packing) of embodiment 3:RNA purification kit
The present embodiment is substantially the same manner as Example 1, and difference is acid processing glass powder and aaerosol solution by 1g:(3-5)
The w/v mixing of ml is made into RNA purified reagent in the same reagent bottle in advance, when in use, appropriate to mix examination
Mixed liquor in agent bottle can be used.
The preparation (mixed packing) of embodiment 4:RNA purification kit
The present embodiment is substantially the same manner as Example 2, and difference is acid processing glass powder and aaerosol solution by 1g:(3-5)
The w/v mixing of ml is made into RNA purified reagent in the same reagent bottle in advance, when in use, appropriate to mix examination
Mixed liquor in agent bottle can be used.
Embodiment 5: RNA Purification Kit blood plasma miRNA is used
The RNA purification kit that the present embodiment is prepared using embodiment 4 carrys out purified plasma miRNA.
The DEPC processing water that the present embodiment is used is prepared as follows: 100 μ L DEPC being added into 100mL ultrapure water, are placed in magnetic
Be stirred overnight on power blender, then using autoclave sterilization pot to mixed liquor the high-temperature process 30min at 120 DEG C, obtain
DEPC handles water.
I. the acquisition of plasma sample
Whole blood 4ml or more is extracted using blood taking needle and anticoagulant tube (containing EDTA), is mixed.Stand 3-4 hours at 4 DEG C (
4 DEG C of refrigerator overnights can be placed).Then 5000rpm is centrifuged 5min at 4 DEG C, and taking supernatant is plasma sample.The plasma sample is not
Anticoagulant heparin can be used, can be reserved in -80 DEG C.
The plasma sample is this specification " sample containing RNA " described above.
II. the extraction of plasma sample
The plasma sample saved at -80 DEG C is placed on ice to melt.Take 250 μ L plasma samples into 1.5ml centrifuge tube.Such as
More miRNA need to be extracted for subsequent experimental, higher volume of sample is can use and be in charge of operation.500 μ L are added into sample
Trizol Reagent is vortexed and mixes 30s, is stored at room temperature 5min.It is another to add preferably to remove the pollutants such as DNA and protein
Enter 200 μ L isopropanols, is vortexed and mixes 30s, be stored at room temperature 5min.Mixed liquor is centrifuged 15min at 4 DEG C, 13000rpm, is shifted
Supernatant is into new 1.5ml centrifuge tube.500 μ L chloroforms are added into supernatant, is vortexed and mixes 10s, be placed at room temperature for 5min.It will
Mixed liquor is centrifuged 15min at 4 DEG C, 13000rpm, shifts supernatant again into new 1.5ml centrifuge tube.Repeat above-mentioned add
Chloroform and centrifugally operated are primary, obtain extracted plasma sample, as this specification " water containing total serum IgE described above
Solution ".
III. the purifying of blood plasma miRNA
Based on the volume of extracted plasma sample, the RNA purification kit prepared from embodiment 4 takes 0.75 times of volume
RNA purified reagent is added in extracted plasma sample, is vortexed and is mixed 20s, places 10min at 4 DEG C.By mixed liquor 4
DEG C, be centrifuged 10min under 6000rpm, careful supernatant of drawing is careful not to be drawn onto bottom glass powder into new 1.5ml centrifuge tube.
The glycogen that the isopropanol and 1 μ L concentration that 0.75 times of volume is added into supernatant are 20mg/ml.Mixed liquor is vortexed and mixes 10s,
It is put at least 2h in -80 DEG C of refrigerators.Centrifuge tube is taken out from -80 DEG C of refrigerators, is placed on ice to melt, after thawing 4 DEG C,
30min is centrifuged under 13000rpm.Liquid carefully is discarded supernatant, is careful not to be drawn onto bottom white precipitate.1ml is added into centrifuge tube
70% ethyl alcohol of pre-cooling is centrifuged 10min at 4 DEG C, 13000rpm after mixing of turning upside down.It repeats to add ethyl alcohol and centrifugation step one
It is secondary, liquid is carefully discarded supernatant, bottom white precipitate is retained.After centrifuge tube brief centrifugation, thoroughly abandoned using small-range pipettor
Remove supernatant, drying at room temperature 2min.12 μ L DEPC are added to centrifugation bottom of the tube and handle water, it is slight to be vortexed after being stored at room temperature 2min
Concussion mixes 5s, that is, extracts and obtain blood plasma miRNA, after detecting, is put into -80 DEG C of refrigerators and saves.
IV. blood plasma miRNA detects
Obtained blood plasma miRNA is extracted above to be detected as follows.
(1) Nanodrop instrument power source is opened, " Nucleic Acids " option is selected；
(2) instrument clean: 1 μ L DEPC processing water is taken to be added in loading wells, close the lid rinse, then uses lens wiping paper
Loading wells and lid are wiped clean；
(3) instrument returns to zero: according to sample solvent type, 1 μ L DEPC processing water being taken to return to zero；
(4) sample detection: taking 1 μ L sample, reads by sample, with lens wiping paper by loading wells and lid after each sample detection
Son is wiped clean, and cleans loading wells and lid with 1 μ L DEPC processing water；
(5) OD260/280, OD260/230 value, the concentration value of each sample are recorded respectively；
(6) with 1 μ L NF-H2O (the silent winged generation that of match) cleaning loading wells and lid, close Nanodrop instrument power source.
(7) gel-dye mix reagent preparation box (Agilent) is taken out, equilibrium at room temperature 30min；
(8) take 550 μ L RNA6000Nano gel matrix into the centrifuge tube with Filter column；
(9) 1500g is centrifuged 10min at room temperature, throws away Filter column；
(10) it takes the 65 filtered gel of μ L, is added 1 μ L dye (carrying out vortex mixing before addition), 13000rpm, room temperature,
It is centrifuged 10min；
(11) 2100 instrument of Agilent and computer software are opened, instrument power source indicator light shows that yellow is normal；
(12) 2100 instrument of Agilent is cleaned:
A) 3 blank chips are taken, mark water, ZAP (the silent winged generation that of match), DEPC respectively,
B) syringe needle 30s first is cleaned without RNase water with 320 μ L,
C) syringe needle 1min is cleaned with 320 μ L ZAP again,
D) syringe needle 2 times finally are cleaned with 320 μ L DEPC water, every time at least 30s,
E) it uncaps and dries syringe needle (be put into before chip to uncap and dry, can not uncap for a long time to prevent pollution)；
(13) moulding:
A) glue pressing device on 2100 instrument of Agilent is modulated into RNA grades (stuck point position is in the first lattice), checks plunger
Air-tightness；
B) prepare new chip, 9 μ L gel-dye mix are added in the hole label " G ", check confirmation bubble-free；
C) lid is covered tightly, is pressed downward plunger until blocking, whole process keeps airtightness；
D) after standing 30s, plunger is unclamped, it is normal for automatically returning at 0.7-0.8mL；
E) 5s is waited, slowly plunger is returned at 1mL；
F) lid is opened, 9.0 μ L gel-dye mix are added to two holes of label " G ".
(14) product are loaded:
A) Marker (to mix before addition) of 5 μ L is respectively added in 13 holes Xiang Qiyu,
B) in the extremely hole of label Marker Ladder after 1 μ L denaturation is added,
C) 12 samples after 1 μ L is denaturalized are sequentially added into the hole sample,
D) chip is vortexed and mixes 1min,
E) sample-adding is completed in 5min, checks in chip there is bubble-free, chip is put into instrument；
(15) operation Agilent 2100 detects program；
(16) after end of run, testing result is saved, detection chip is taken out, cleans syringe needle without the water of RNA enzyme with 320 μ L
1min dries, and closes the lid, and closes power supply.
V. blood plasma miRNA sample quality standard
Since the blood plasma miRNA sample of extraction is mainly used for high-throughput sequencing library building, formulated such as subsequent use
Lower sample quality standard.
(1) miRNA sample itself is slight sticky without obvious sediment or pigment；
(2) in Nanodrop testing result, miRNA sample concentration is greater than 5ng/ μ L, OD260/280 between 1.5-3,
OD260/230 is generally less than 2；
(3) in 2100 testing result of Agilent, baseline is steady, and 200bp or more has peak without obvious miscellaneous peak, 200bp or less
Type occurs；
(4) sample extraction quality constructs result with high-throughput sequencing library and sequencing data analysis result is final judgement mark
It is quasi-.
VI. the comparison of the sequencing library building result of different blood plasma miRNA method for extraction and purification
For verify the present embodiment blood plasma miRNA method for extraction and purification effect, to same plasma sample, using routine
QIAamp CircuLating Nucleic Acid Kit, mirVanaTMPARISTMKit and Trizol method presses kit
Specification extracts purifying, and is compared with the blood plasma miRNA method for extraction and purification of the present embodiment, as a result such as table 1 and Fig. 1
It is shown.
Table 1: the comparison of the sequencing library building result of different blood plasma miRNA method for extraction and purification
Seen from table 1, using Trizol method for extraction and purification, library inspection result is unqualified, using QIAamp
CircuLating Nucleic Acid Kit and mirVanaTMPARISTMKit method for extraction and purification, library inspection result is qualified but has
Miscellaneous band, and the method for extraction and purification of the present embodiment is used, it is qualified that result is examined in library.From left to right, the first band is that the present invention is real to Fig. 1
That applies example builds library as a result, the single concentration of library band, and brightness is high, second and third strip be all Marker, be
Quick-Load pBR322DNA-MspI Digest standard items in the reagent preparation box of Small RNA multisample library, the 4th
Band is that QIAamp CircuLating Nucleic Acid Kit extracts the library of product building as a result, Article 5 band is
mirVanaTMPARISTMKit extracts the result in product building library.Therefore, as table 1 and Fig. 1 as it can be seen that representated by the present embodiment
Blood plasma miRNA method for extraction and purification effect of the present invention is best.
VII. the sequencing data quality of the blood plasma miRNA extraction purification product of the present embodiment
The blood plasma miRNA product obtained using the present embodiment extraction purification, in high-flux sequence instrument (Illumina Hiseq
2500) it is sequenced on.Table 2 shows the sequencing data quality of the blood plasma miRNA extraction purification Product samples of the present embodiment, figure
2 show the sequence length distribution of one of Product samples.
Table 2: the sequencing data quality of the blood plasma miRNA extraction purification product of the present embodiment
As can be seen from Table 2, the sample of test repeatedly extracts the miRNA obtained three times by this method, under constructed library
In machine data, valid data ratio 97% or more, illustrates this extracting method operation repeatability with higher, same to eight-legged essay
Library sequencing quality is high.From Figure 2 it can be seen that lower machine data Insert Fragment is sequenced and concentrates, mainly exists by the miRNA that this method is extracted
21nt or so meets the description that each document is distributed miRNA segment.For synthesis, the miRNA extracted by this method,
Segment intensity is high, and literature data quality and data effective percentage all reach reasonable level.
Embodiment 6: RNA Purification Kit serum miRNA is used
The present embodiment and the operation of embodiment 5 are basically the same, and only the acquisition method of serum sample is different.Specifically
Ground, serum sample can be acquired as follows.
Using blood taking needle, 4ml or more blood is acquired.Whole blood is instilled to clean EP pipe or 15ml centrifuge tube.By whole blood
3-4 hours are stood at 4 DEG C, it is seen that (can also be placed in 4 DEG C of refrigerator overnights) is precipitated in clot.By the whole blood after placement 4 DEG C,
10min is centrifuged under 5000rpm, it is seen that faint yellow serum.Aspirate supernatant into new centrifuge tube, then by supernatant 4 DEG C,
10min, Aspirate supernatant to new centrifuge tube, utmostly to guarantee Serology Quality are centrifuged under 3000rpm.Serum is dispensed,
It is stored in -80 DEG C.
The serum sample collected is this specification " sample containing RNA " described above.It can refer to embodiment 5
Method purify to obtain serum miRNA.
Use above specific example is expounded the present invention, is merely used to help understand the present invention, not to
The limitation present invention.The design of those skilled in the art according to the present invention can also be made and several simply push away
It drills, deform or replaces.These are deduced, deformation or alternative are also fallen into scope of the presently claimed invention.
Claims (10)
1. a kind of RNA purification kit, which is characterized in that the RNA purification kit includes acid processing glass powder and suspends molten
Liquid, the acid processing glass powder have 325 mesh or thinner granularity, and the aaerosol solution includes 0.2- using ultrapure water as solute
The chaotropic salt of 1M and the buffer of 0.05-0.2M, pH 5-7.
2. RNA purification kit described in claim 1, which is characterized in that the chaotropic salt is lithium chloride, potassium rhodanide, height
Sodium chlorate or their combination.
3. RNA purification kit described in claim 1, which is characterized in that the buffer be ammonium chloride, disodium hydrogen phosphate,
Sodium dihydrogen phosphate, sodium acetate or their combination.
4. RNA purification kit described in claim 1, which is characterized in that the aaerosol solution also includes 0.05-2mM's
EDTA。
5. RNA purification kit according to claim 4, which is characterized in that the aaerosol solution includes the chlorination of 0.5M
The EDTA of lithium, the ammonium chloride of 0.1M and 1mM.
6. RNA purification kit according to any one of claims 1-5, which is characterized in that the acid processing glass powder
It is divided in the aaerosol solution in different reagent bottles.
7. RNA purification kit according to any one of claims 1-5, which is characterized in that the acid processing glass powder
Formation RNA purified reagent in the same reagent bottle, the acid processing glass powder and the suspension are blended in the aaerosol solution
The w/v of solution is 1g:(3-5) ml.
8. a kind of method for being enriched with miRNA from the aqueous solution containing total serum IgE, which is characterized in that the described method includes:
(1) aqueous solution containing total serum IgE is provided；
(2) acid in RNA purification kit according to claim 6 is handled into glass powder and the aaerosol solution mixes
It closes now to match RNA purified reagent, wherein the w/v of the acid processing glass powder and the aaerosol solution is 1g:(3-5)
ml；Or provide the RNA purified reagent of RNA purification kit according to claim 7 prepared；
(3) the RNA purified reagent of 0.6-1 times of volume is added into the aqueous solution containing total serum IgE, mixes, obtains the
One mixed liquor is placed 5-15 minutes at 4 DEG C；
(4) first mixed liquor is centrifuged 5-15 minutes at 4 DEG C, 5000-8000rpm, takes supernatant, be added 0.75-1 times
The isopropanol of volume, and 10-20 μ g glycogen is added, obtain the second mixed liquor；
(5) second mixed liquor is placed at least 2h at -80 DEG C, then melted, is centrifuged at 4 DEG C, 10000-15000rpm
30-40 minutes, discard supernatant liquid；
(6) 70% ethyl alcohol of pre-cooling is added to resulting sediment, mixes, is centrifuged 10-15 at 4 DEG C, 10000-15000rpm
Minute, discard supernatant liquid；
(7) step (6) are repeated once, discards supernatant liquid, bottom is had into the centrifuge tube of sediment wink at 4000-6000rpm
When centrifugation 5s after, thoroughly discard supernatant liquid using small-range pipettor, drying at room temperature 2-5 minute, obtain being enriched sinking for miRNA
Starch；
(8) optionally, suitable pyrocarbonic acid diethyl ester (DEPC) processing water, room is added in the sediment that Xiang Suoshu is enriched miRNA
After temperature stands 2-5 minutes, mixes, obtain the aqueous solution for being enriched miRNA.
9. preparation method according to claim 8, which is characterized in that the aqueous solution of the offer containing total serum IgE include:
(A) sample containing RNA is provided；
(B) the Trizol Reagent of 2-2.5 parts by volume is added to the sample described in 1 parts by volume containing RNA, mixes, room temperature is quiet
It sets 5-10 minutes；
(C) isopropanol of 0.8-1 parts by volume is added into step (B) resulting mixed liquor, mixes, is stored at room temperature 5-10 minutes；
(D) step (C) resulting mixed liquor is centrifuged 10-15 minutes at 4 DEG C, 10000-15000rpm, takes supernatant to new
Centrifuge tube in；
(E) chloroform of 2-2.5 parts by volume is added to the gained supernatant of 1 parts by volume, mixes, is stored at room temperature 5-10 minutes；
(F) step (E) resulting mixed liquor is centrifuged 10-15 minutes at 4 DEG C, 10000-15000rpm, takes supernatant to new
Centrifuge tube in；
(G) it repeats step (E) and (F) once, obtains the aqueous solution containing total serum IgE.
10. preparation method according to claim 9, which is characterized in that the sample containing RNA is blood plasma or serum.
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