CN1189474C - Method of separating whole length, complete messenger RNA from total RNA - Google Patents
Method of separating whole length, complete messenger RNA from total RNA Download PDFInfo
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Abstract
The present invention relates to a method for effectively separating complete full-length messenger ribonucleic acid (mRNA) from total ribonucleic acid (total RNA). The present invention is characterized in that sodium dodecyl sulfate or sodium lauryl sulfonate is used as a ribonuclease (RNase) inhibitor and is added to a solution for dissolving the total RNA and a solution system for separating the mRNA for preventing the degradation of the mRNA by the RNase. Moreover, two kinds of buffer solutions are used for washing and removing other kinds of RNA except the mRNA and possibly existing RNase activities to effectively separate out complete full-length mRNA molecules. The method of the present invention has the advantages of low cost and high mRNA yield, and is especially suitable for separating and purifying the mRNA from the total RNA extracted from endogenous mammalian cells or tissues with high RNase activities.
Description
Technical field
The present invention relates to a kind of method of from total Yeast Nucleic Acid, effectively separating total length, complete messenger RNA(mRNA).
Technical background
In the research of molecular biology and molecular genetics, although many analysis and research that relate to Yeast Nucleic Acid (RNA) can be made of total RNA sample, such as making up reverse transcription thymus nucleic acid (cDNA) library, detecting rare messenger RNA(mRNA) (mRNA), S
1Must adopt high-quality total length, complete mRNA sample in the research of nuclease mapping, external translation etc.Therefore, the separation and purification of mRNA be engaged in molecular biology research one of the basic skills that must grasp.
In eukaryotic total RNA, rRNA (rRNA), transfer ribonucleic acid (tRNA) etc. has accounted for more than 95%, and mRNA only accounts for 1～5%.Because 3 of most eukaryotic mRNA ' end all has polyadenylic acid [Poly (the A)] tail end that is uneven in length, can form hydrogen bond with oligomerization deoxythymidine [Oligo (dT)], thereby can be with Oligo (dT) Mierocrystalline cellulose affinity chromatography with mRNA separation and purification (the Sambrook J that comes out, et al.Molecular Cloning:aLaboratory Manual.2nd ed..New York:Cold Spring Harbor Laboratory Press, 1989.).
At first set up (Aviv H in 1972 with Oligo (dT) Mierocrystalline cellulose affinity chromatography separating mRNA by Aviv and Leder, Leder P..Purification of biolgically active globin messenger RNA by chromatographyon oligothmisylic acid-cellulose.Proc Natl.Acad.Sci.U.SA., 1972,69:1408-1412.), although afterwards this basic skills was done some little modifications, its ultimate principle and elementary operation do not change.
At present the separation method of mRNA has 2 kinds: a kind of is direct separating mRNA from the lysate of biological tissue or cell, and another kind is a separating mRNA from total RNA sample.
MRNA is subject to rnase (RNase) degraded, and RNase very " stubbornness " be difficult for deactivation.Therefore to from total RNA sample, isolate high-quality total length, complete mRNA, necessary:
(1) agents useful for same, solution, apparatus etc. are carried out strict RNase inactivation treatment;
(2) carefully avoid the pollution of exogenous RNase in operation to reagent, solution, apparatus etc.;
(3) the active work of endogenous RNase residual in total RNA sample effectively suppressed or inactivation treatment.
The supply of various mRNA separating kit is arranged in the market, the principal feature of these test kits is to replace fibrous Oligo (dT) Mierocrystalline cellulose with particulate Oligo (dT) sorbent material, replace traditional column chromatography (Chromatography column protocol) with column spinner method (Spin-column protocol), and be user's a complete set of reagent and material of using of separating mRNA all set, thereby simplified operation steps, shorten disengaging time, improved working efficiency.For example the sorbent material used of the mRNA separating kit of Qiagen company is that Oligo (dT) is connected on the polystyrene micelle, becomes little pearl, and commodity are called Oligotex.The separation method of this test kit is: (1) dissolving: with the total RNA of water dissolution of no RNase; (2) sex change: add the adsorption-buffering liquid of forming by 2mmol/L Tutofusin tris (Tris)-hydrochloric acid (HCl) of pH 7.5 and 2mmol/L ethylenediamine tetraacetic acid (EDTA) (ETDA) and 1mol/L NaCl and 2g/L sodium lauryl sulphate, add sorbent material Oligotex again, then in 70 ℃ of water bath processing 3 minutes; (3) absorption: from 70 ℃ of water-baths, take out the sex change sample, put and left standstill under the room temperature 10 minutes; (4) washing: use the lavation buffer solution of forming by the 10mmol/L Tris-HCl of pH 7.5 and lmmol/L EDTA and 0.15mol/LNaCl to wash Oligotex 2 times; (5) wash-out: with the 5mmol/LTris-HCl elution buffer that is preheated to 70 ℃ pH 7.5, wash-out is adsorbed on the mRNA (Oligotex on the Oligotex
TMHandbook.Qiagen, July1999.).
Although test kit brings many convenience for the mRNA mask work, still have weak point: (1) mRNA separation yield is not high; (2) cost an arm and a leg, this laboratory for need separating mRNA in enormous quantities is difficult to bear.
In addition, the present total RNA separation method that generally adopts, for example TRIZOL method and " single stage method " (ChomzynskiP., Sacchi N..Single-step method of RNA isolation by acid guanidiniumthiocyanate-phenol-chloroform extraction.Anal.Biochem., 1987,162:156-159.) etc., usually residual in total RNA that it is separated to have endogenous RNase activity, thereby usually cause the isolating failure of mRNA.Therefore, need a kind of mRNA yield height, with low cost and can effectively suppress and eliminate the endogenous RNase activity that may exist in total RNA sample in the present technique field, guarantee to be separated to the method for total length, complete mRNA.
Summary of the invention
The object of the present invention is to provide a kind of mRNA yield height, with low cost, and can effectively suppress and eliminate the endogenous RNase activity in total RNA sample, guarantee to be separated to the method for total length, complete mRNA.
Technical characterictic of the present invention is: (1), is used in total RNA dissolving and isolating each step of mRNA as the RNase inhibitor with sodium lauryl sulphate or sodium laurylsulfonate; (2) be in the suds and used two kinds of damping fluids that are added with sodium lauryl sulphate or sodium laurylsulfonate, more up hill and dale flush away other RNA beyond the mRNA, improved the purity of mRNA, and removed the RNase activity that may exist effectively, thereby can use cheap Oligo (dT) Mierocrystalline cellulose to make sorbent material, reach high yield, from total RNA sample, successfully separate total length, complete mRNA at low cost, realized purpose of the present invention.
A kind of method of from total Yeast Nucleic Acid (english abbreviation Total RNA), effectively separating total length, complete messenger RNA(mRNA) (english abbreviation mRNA) of the present invention, comprise: (1) dissolving, (2) sex change, (3) absorption, (4) washing, (5) 5 steps of wash-out in order more to clearly demonstrate the technology of the present invention feature, describe in detail 5 steps respectively below.
(1) dissolving: total Yeast Nucleic Acid dry product is dissolved in the water of the deoxyribonuclease that contains 0.5～5g/L sodium lauryl sulphate or sodium laurylsulfonate, measure the concentration of total Yeast Nucleic Acid in the water then, and detect the quality of total Yeast Nucleic Acid with the sex change agarose gel electrophoresis, when observe 18S rRNA (english abbreviation rRNA) band and 28S rRNA be with clear, and both luminance factors are 1: 2 o'clock, then can carry out next step.
The content of described sodium lauryl sulphate or sodium laurylsulfonate is preferably 1g/L, described sex change agarose gel electrophoresis detects can adopt normally used method, for example the detection of denaturing formaldehyde agarose gel electrophoresis or oxalic dialdehyde-dimethyl sulfoxide (DMSO) sex change agarose gel electrophoresis detection method etc.
(2) sex change: the qualified total Yeast Nucleic Acid aqueous solution of above-mentioned detection is placed in 60～70 ℃ of water-baths handled 3～25 minutes, to destroy the secondary structure of Yeast Nucleic Acid, make its polyadenylic acid [english abbreviation Poly (A)] tail end expose out, put immediately in the bath of ice bath or cryosel and be cooled to 4～10 ℃.
Preferably 65 ℃ of described bath temperatures, in preferably 20 minutes treatment time, cooling time, requirement was not very strict, according to the temperature of cooling bath and different, as long as be cooled to 4～10 ℃.
(3) absorption: in above-mentioned process total rna solution of sex change, add isopyknic with it adsorption-buffering liquid, sorbent material adds in the ratio of the total Yeast Nucleic Acid 3～150mg of 1mg weight in wet base (preferably 120mg weight in wet base) then, adsorbs messenger RNA(mRNA) in total Yeast Nucleic Acid with affinity chromatography.
Described adsorption-buffering liquid consists of 5～100mmol/L Tutofusin tris-hydrochloric acid (being Tris-HCl) and 1～10mmol/L ethylenediamine tetraacetic acid (EDTA) (being EDTA) and 0.8～1.2mol/L sodium-chlor (being NaCl) and 0.5～5g/L sodium lauryl sulphate or the sodium laurylsulfonate of pH 7.0～8.0, and best composition is 20mmol/L Tutofusin tris-hydrochloric acid and 2mmol/L ethylenediamine tetraacetic acid (EDTA) and 1mol/L sodium-chlor and 1g/L sodium lauryl sulphate (being english abbreviation SDS) or the sodium laurylsulfonate of pH 7.5.
Described sorbent material is the covalently bound material that forms on carrier of oligomerization deoxythymidine [english abbreviation Oligo (dT)], for example the oligomerization deoxythymidine is connected and becomes fibrous on the Mierocrystalline cellulose, be that commodity cellulosic material of Oligo (dT) by name or oligomerization deoxythymidine are connected on the polystyrene micelle, become little pearl, the material of commodity Oligotex by name etc., described sorbent material can also have the same function of oligomerization deoxythymidine, can be connected the material that forms on the carrier with the polyadenylic acid of the messenger RNA(mRNA) 3 ' end material by hydrogen bonded, polyuridylic acid-dextrane gel for example, the i.e. material of commodity Poly (U) by name-Sephadex.
Described affinity chromatography is normally used method, for example partition method, column spinner method, column chromatography etc., also partition method and the coupling of column spinner method in batches in batches.
Described absorption can be adopted standing adsorption, also can the level vibration adsorb, and for example can adopt under the room temperature with 50～80r/min level vibration 30～60 minutes.
(4) washing: after above-mentioned adsorption step is finished, divide the liquid phase of leaving away, the sorbent material of solid phase washs with level pad earlier, and then washs with lavation buffer solution, to remove other Yeast Nucleic Acid beyond the messenger RNA(mRNA) and the RNase activity that may exist on the sorbent material, the liquid phase of leaving away is divided in the washing back.
The composition of described level pad is 5～100mmol/L Tutofusin tris-hydrochloric acid and 0.5～10mmol/L ethylenediamine tetraacetic acid (EDTA) and 0.4～0.6mol/L sodium-chlor and 0.5～5g/L sodium lauryl sulphate or the sodium laurylsulfonate of pH 7.0～8.0, and best composition is 10mmol/L Tutofusin tris-hydrochloric acid and 1mmol/L ethylenediamine tetraacetic acid (EDTA) and 0.5mol/L sodium-chlor and 1g/L sodium lauryl sulphate or the sodium laurylsulfonate of pH 7.5.
The composition of described lavation buffer solution is: 5～100mmol/L Tutofusin tris-hydrochloric acid of pH 7.0～8.0 and 0.5～10mmol/L ethylenediamine tetraacetic acid (EDTA) and 0.1～0.2mol/L sodium-chlor and 0.5～5g/L sodium lauryl sulphate or sodium laurylsulfonate, best composition are 10mmol/L Tutofusin tris-hydrochloric acid and 1mmol/L ethylenediamine tetraacetic acid (EDTA) and 0.15mol/L sodium-chlor and 1g/L sodium lauryl sulphate or the sodium laurylsulfonates of pH 7.5.
The consumption of level pad and lavation buffer solution and washing times do not have strict restriction, consumption has lacked can be washed several times more, consumption is many can be washed several times less, consumption is more, washing times is more, also can, but consumption is too many or washing times is too many, both waste solvent and also lost time, also may cause the loss of mRNA.Preferably, wash 2～3 times in the ratio of 1mg total Yeast Nucleic Acid 0.5～1mL level pad or lavation buffer solution.
The leave away method of liquid phase of described branch is to adopt usual method, for example centrifugal, post, filtration etc. excessively, according to used affinity chromatography difference, can adopt diverse ways, can adopt centrifugation to go liquid phase when for example adopting column spinner method or batch partition method or batch partition method and column spinner method bonded method.
(5) wash-out: with room temperature to 70 ℃, preferably 65 ℃ elution buffer comes out the messenger RNA(mRNA) wash-out that is adsorbed on the sorbent material, collects the elution buffer that contains messenger RNA(mRNA) then.
The composition of described elution buffer is: 5～20mmol/L Tutofusin tris-hydrochloric acid of pH 7.0～8.0 and 0～5mmol/L ethylenediamine tetraacetic acid (EDTA) and 0～1g/L sodium lauryl sulphate or sodium laurylsulfonate, best composition are 10mmol/L Tutofusin tris-hydrochloric acid and the 1mmol/L ethylenediamine tetraacetic acid (EDTA)s of pH7.5.
The consumption of elution buffer and wash-out number of times be according to used affinity chromatography and different, and be to batch partition method and the column spinner method ratio in 1mg Yeast Nucleic Acid 60～100 μ L elution buffers, more to the column chromatography consumption.Best 2～3 times of the number of times of wash-out.The collection method that contains the messenger RNA(mRNA) elutriant adopts usual method, can cross post and separate, can centrifugation, and also different because of used chromatography.
Total Yeast Nucleic Acid of the present invention can be from (1) tissue or cell, (2) animal tissues or cell, (3) plant tissue or cell, (4) other eukaryote tissue or cell are that " single stage method " or other separation method precipitation obtain by TRIZOL reagent method or acidity-guanidine thiocyanate-phenol-chloroform method.
Water that the present invention is used and solution except that Tutofusin tris (english abbreviation Tris) solution, all need add baycovin (english abbreviation DEPC) to 1g/L processing and spend the night, then autoclaving.Tris solution need be prepared with Tris, water and the container of no RNase, then autoclaving.
The used sorbent material of the present invention need suspend with the NaOH of 0.1mol/L and wash centrifugal 5～7 times, and suspend with level pad then and wash centrifugal 5～7 times, be 7.5 until supernatant liquor pH.
Above-mentioned requirement and treatment process to institute of the present invention water, reagent, container and sorbent material all is basic requirement of this area experimental implementation and method, is not peculiar technology of the present invention.
A kind of method of from total Yeast Nucleic Acid, effectively separating total length, complete messenger RNA(mRNA) of the present invention, owing to adopted following feature technology: (1) has all used sodium lauryl sulphate or the sodium laurylsulfonate inhibitor as RNase in the sepn process of total RNA dissolving, mRNA absorption, washing, wash-out, effectively suppressed the endogenous RNase activity that may exist in the RNA sample, made it can not damage mRNA; (2) two kinds of damping fluids that are added with sodium lauryl sulphate or sodium laurylsulfonate in washing process, have been adopted, at first use level pad, under this solution condition, mRNA can firmly be retained on the sorbent material, and other RNA wash-out beyond the mRNA is come out, then use lavation buffer solution, mRNA other RNA wash-out in addition with the sorbent material non-specific binding is come out, improved the isolating purity of mRNA like this, and removed the RNase activity that may exist, make the present invention can adopt less expensive oligomerization deoxythymidine Mierocrystalline cellulose like this, be the cellulosic sorbent material of trade(brand)name Oligo (dT), realize from total RNA, effectively isolating total length, complete mRNA.
The present invention has following advantage:
(1) with low cost: separation costs only is 3.4～5.0 yuan/μ gmRNA;
(2) mRNA yield height: mRNA separates yield and is stabilized in the total RNA of 20～50 μ g/mg, because of the total RNA sample from different tissues different;
(3) quality-guarantee: can effectively suppress the endogenous RNase activity that may exist in total RNA sample, guarantee to be separated to total length, complete mRNA, and good reproducibility, the mRNA of gained can directly or do to be used for NorthernBlots hybridization analysis, reverse transcription-polymerase chain reaction (RT-PCR), S behind the suitable purifying
1Restriction analysis, external translation etc.;
(4) applied widely: as to can be used for the separation of different types of eukaryotic mrna, separation and purification mRNA among the total RNA that is specially adapted to from active high mammalian tissues of endogenous RNase or cell, extract.
The separating effect of the mRNA separating kit of the present invention and Qiagen company relatively sees the following form:
| Separating effect | The present invention | The mRNA separating kit of Qiagen company |
| 1.mRNA integrity | Separable to total length, complete mRNA | Separable to total length, complete mRNA |
| 2.mRNA yield | The total RNA of 24～50 μ g/mg | The total RNA of 16～30 μ g/mg |
| 3.mRNA hybridization signal | By force | {。##.##2}, |
| 4. separation costs | 3.4～5.0 yuan/μ g mRNA | 15.0～28.0 yuan/μ g mRNA |
Description of drawings
Fig. 1:
The mRNA electrophoretogram of mouse (2 μ g/ swimming lane);
M: molecular mass standard;
MRNA source: 1. brain, the 2. heart, 3. liver, 4. lung, 5. spleen, 6. kidney, 7. testis, 8. skeletal muscle.
Fig. 2:
Mouse mRNA Northern Blots is hybridized collection of illustrative plates (mRNA 2 μ g/ swimming lanes);
MRNA source: 1. brain, the 2. heart, 3. liver, 4. lung, 5. spleen, 6. kidney, 7. testis, 8. skeletal muscle.
β-actin probe (8ng/mL) results of hybridization with the DIG mark.
Fig. 3:
The RT-PCR G3PDH gene fragment (1.2kb) that increases;
With Mouse Liver mRNA is masterplate, the masterplate consumption: 1. 905ng; 2. 452ng;
3.?181ng；4.?90ng；5.?18ng。
Fig. 4:
People's mRNA electrophoretogram (2 μ g/ swimming lane);
M: molecular mass standard;
MRNA source: 1. brain, the 2. heart, 3. liver, 4. lung, 5. spleen, 6. stomach, 7. testis, 8. skeletal muscle.
Fig. 5:
People mRNA Northern Blots is hybridized collection of illustrative plates (mRNA 2 μ g/ swimming lanes);
MRNA source: 1. brain, the 2. heart, 3. liver, 4. lung, 5. spleen, 6. stomach, 7. testis, 8. skeletal muscle.
β-actin probe (8ng/mL) results of hybridization with the DIG mark.
Fig. 6:
The RT-PCR p53 gene fragment (1.0kb) that increases;
People liver mRNA is a masterplate, the masterplate amount: 1. 30ng, 2. 185ng, 3. 925ng.
Embodiment
The following examples are to illustrate the present invention, rather than limit.
Embodiment 1: separation and purification mRNA from the isolating total RNA of mouse tissue.
Required reagent of A and material
1. total RNA of 8 kinds of tissues of mouse (brain, the heart, liver, lung, spleen, kidney, testis, skeletal muscle) (separating) with " single stage method ".
2. there is not RNase water (being added with 1g/L SDS).
3. the 20mmol/L Tris-HCl of adsorption-buffering liquid: pH 7.5 and 2mmol/L EDTA and 1mol/L NaCl and 1g/L SDS.
4. the 10mmol/L Tris-HCl of level pad: pH 7.5 and 1mmol/L EDTA and 0.5mol/L NaCl and 1g/L SDS.
5. the 10mmol/L Tris-HCl of lavation buffer solution: pH 7.5 and lmmol/L EDTA and 0.15mol/L NaCl and 1g/L SDS.
6. the 10mmol/L Tris-HCl of elution buffer: pH 7.5 and 1mmol/L EDTA.
Illustrate: above-mentioned solution all need add DEPC (diethylpyrocarbonate) and handle to 1g/L and spend the night, then autoclaving except that Tris.Tris solution need be prepared with Tris, water and the container of no RNase, then autoclaving.
7.Oligo (dT)-and Cellulose Type 7 (Pharmacia company product): take by weighing 320 milligrams of Oligo (dT) Mierocrystalline cellulose, adding 0.1mol/L NaOH soaked 10～20 minutes, 12000r/min removed supernatant in centrifugal 3 minutes, suspend with 0.1mol/L NaOH again and wash centrifugal 5 times, suspend with level pad then and wash centrifugal 7 times, until supernatant pH is 7.5, at last that Oligo (dT) cellulose suspension is standby in level pad.
Column spinner (MSK-100, Axygen).
The B experimental implementation
1. will precipitate the dry total RNA of mouse that comes out is dissolved in the no RNase water that is added with SDS, the sampling and measuring total rna concentration, and carry out the quality of the total RNA of denaturing formaldehyde agarose gel electrophoresis inspection, if up-to-standard (it is clear that 18SrRNA and 28SrRNA are with, and luminance factor is 1: 2) then can be used for the mRNA separation, on the contrary then unavailable.
2. get the total RNA of 500 μ L (concentration 2mg/mL) in the 1.5mL centrifuge tube, put in 65 ℃ of water-baths to handle and made the RNA sex change in 20 minutes, put in the ice bath cooling then immediately 10 minutes.
3. add 500 μ L adsorption-buffering liquid mixings, press amount adding Oligo (dT) Mierocrystalline cellulose that the total RNA of 1mg adds the 120mg weight in wet base, (50～80r/min) the 60 minutes absorption mRNA of level vibration at room temperature.
4. under the room temperature, 12000r/min removed supernatant in centrifugal 3 minutes.
5. add 0.7mL level pad suspension washing Oligo (dT) Mierocrystalline cellulose, 12000r/min removed supernatant in centrifugal 3 minutes.Wash altogether 3 times.
6. add 0.7mL lavation buffer solution suspension washing 2 times again, 12000r/min removed supernatant in centrifugal 3 minutes.
7. with the 0.7mL lavation buffer solution Oligo (dT) Mierocrystalline cellulose all is washed till in the column spinner, 10000r/min removed lavation buffer solution in centrifugal 1 minute.
8. column spinner is transferred in another clean 1.5mL pipe, in post, adds 80 μ L elution buffers (65 ℃ of preheatings), left standstill 1 minute, at 10000r/min centrifugal 30 seconds, collect the mRNA that wash-out comes out.
9. add 80 μ L elution buffers (65 ℃ of preheatings) again and in pillar, carry out the wash-out second time.
10. the mRNA that 2 wash-outs are come out merges, and puts-70 ℃ of preservations.
11. result:
A. detect through the denaturing formaldehyde agarose gel electrophoresis and show, the size of 8 kinds of mouse tissue mRNA that are separated to the inventive method mainly concentrates in 0.5～8.0kb (kilobase to) scope, no signs of degradation (see figure 1).
B. β-actin the probe with the DIG mark carries out Northern Blots hybridization analysis, and the result shows the clear sharp keen (see figure 2) of β-actin hybrid belt, illustrates that the mRNA that the present invention is separated to is a total length, complete.
C. carry out the amplification of G3PDH Gene RT-PCR with the Mouse Liver mRNA that is separated to as masterplate and obtain the good result (see figure 3), illustrate that the isolated mRNA of the present invention is complete, quality is high.
Embodiment 2: separation and purification mRNA from the isolating total RNA of tissue.
Required reagent of A and material
1. total RNA of 8 kinds of tissues of human body (brain, the heart, liver, lung, spleen, stomach, testis, skeletal muscle) (separating) with TRIZOL reagent.
2. there is not RNase water (being added with 1g/L SDS).
3. the 20mmol/L Tris-HCl of adsorption-buffering liquid: pH 7.5 and 2mmol/L EDTA and 1mol/L NaCl and 1g/L SDS.
4. the 10mmol/L Tris-HCl of level pad: pH 7.5 and 1mmol/L EDTA and 0.5mol/L NaCl and 1g/L SDS.
5. the 10mmol/L Tris-HCl of lavation buffer solution: pH 7.5 and 1mmol/L EDTA and 0.15mol/L NaCl and 1g/L SDS.
6. the 10mmol/L Tris-HCl of elution buffer: pH 7.5 and 1mmol/L EDTA.
Illustrate: above-mentioned solution all need add DEPC (diethylpyrocarbonate) and handle to 1g/L and spend the night, then autoclaving except that Tris.Tris solution need be prepared with Tris, water and the container of no RNase, then autoclaving.
7.Oligo (dT)-and Cellulose Type 7 (product of Pharmacia company): take by weighing 320 milligrams of Oligo (dT) Mierocrystalline cellulose, adding 0.1mol/L NaOH soaked 10～20 minutes, 12000r/min removed supernatant in centrifugal 3 minutes, suspend with 0.1mol/L NaOH again and wash centrifugal 5 times, suspend with level pad then and wash centrifugal 7 times, until supernatant pH is 7.5, at last that Oligo (dT) cellulose suspension is standby in level pad.
Column spinner (MSK-100, Axygen).
The B experimental implementation
1. will precipitate the dry total RNA of human body that comes out and be dissolved in the no RNase water that is added with SDS, the sampling and measuring total rna concentration, and carry out the denaturing formaldehyde agarose gel electrophoresis and detect total RNA quality.If then can be used for mRNA, separates total RNA up-to-standard (it is clear that 18SrRNA and 28SrRNA are with, and luminance factor is 1: 2), on the contrary then unavailable.
2. get the total RNA of 500 μ L (concentration 2mg/mL) in the 1.5mL centrifuge tube, put in 65 ℃ of water-baths to handle and made the RNA sex change in 20 minutes, put in the ice bath cooling then immediately 10 minutes.
3. add 500 μ L adsorption-buffering liquid mixings, press amount adding Oligo (dT) Mierocrystalline cellulose that the total RNA of 1mg adds the 120mg weight in wet base, (50～80r/min) the 60 minutes absorption mRNA of level vibration at room temperature.
4. under the room temperature, 12000r/mim removed supernatant in centrifugal 3 minutes.
5. add 0.7mL level pad suspension washing Oligo (dT) Mierocrystalline cellulose, 12000r/min removed supernatant in centrifugal 3 minutes.Wash altogether 3 times.
6. add 0.7mL lavation buffer solution suspension washing 2 times again, 12000r/min removed supernatant in centrifugal 3 minutes.
7. with the 0.7mL lavation buffer solution Oligo (dT) Mierocrystalline cellulose all is washed till in the column spinner, 10000r/min removed lavation buffer solution in centrifugal 1 minute.
8. column spinner is transferred in another clean 1.5mL pipe, adds 80 μ L elution buffers (65 ℃ of preheatings), left standstill 1 minute, at 10000r/min centrifugal 30 seconds, collect the mRNA that wash-out comes out.
9. add 80 μ L elution buffers (65 ℃ of preheatings) again and in pillar, carry out the wash-out second time.
10. the mRNA that 2 wash-outs are come out merges, and puts-70 ℃ of preservations.
11. result:
A. detect through the denaturing formaldehyde agarose gel electrophoresis and show, the size of 8 kinds of tissue mRNA that are separated to the inventive method mainly concentrates in 0.5～8.0kb scope no signs of degradation (see figure 4).
B. β-actin the probe with the DIG mark carries out Northern Blots hybridization analysis, and the result shows the clear sharp keen (see figure 5) of β-actin hybrid belt, illustrates that the mRNA that is separated to is a total length, complete.
C. carry out the amplification of p53 Gene RT-PCR with the people liver mRNA that is separated to as masterplate and obtain the good result (see figure 6), illustrate that the isolated mRNA of the present invention is complete, quality is high.
Claims (3)
1, a kind of method of effectively separating total length, complete messenger RNA(mRNA) from total Yeast Nucleic Acid comprises 5 steps:
(1) dissolving: total Yeast Nucleic Acid dry product is dissolved in the water of the deoxyribonuclease that contains 0.5～5g/L sodium lauryl sulphate or sodium laurylsulfonate, measure total Yeast Nucleic Acid concentration in the water then, and detect the quality of total Yeast Nucleic Acid with the sex change agarose gel electrophoresis, when observing 18S rRNA band and 28S rRNA band is clear, and both luminance factors are 1: 2 o'clock, then can carry out next step;
(2) sex change: the qualified total Yeast Nucleic Acid aqueous solution of above-mentioned detection is put into 60～70 ℃ of water-baths handled 3～25 minutes, to destroy the secondary structure of Yeast Nucleic Acid, make its polyadenylic acid tail end expose, put immediately in the bath of ice bath or cryosel and be cooled to 4～10 ℃;
(3) absorption: in above-mentioned process total rna solution of sex change, add isopyknic with it adsorption-buffering liquid, sorbent material adds in the ratio of the total Yeast Nucleic Acid 3～150mg of 1mg weight in wet base then, adsorbs messenger RNA(mRNA) in total Yeast Nucleic Acid with affinity chromatography; Described adsorption-buffering liquid consists of 5～100mmol/L Tutofusin tris-hydrochloric acid and 1～10mmol/L ethylenediamine tetraacetic acid (EDTA) and 0.8～1.2mol/L sodium-chlor and 0.5～5g/L sodium lauryl sulphate or the sodium laurylsulfonate of pH 7.0～8.0, described sorbent material be the oligomerization deoxythymidine covalently bound the material that forms on the carrier or have the same function of oligomerization deoxythymidine, can be connected the material that forms on the carrier with the polyadenylic acid of the messenger RNA(mRNA) 3 ' end material by hydrogen bonded;
(4) washing: after above-mentioned adsorption step is finished, divide the liquid phase of leaving away, the sorbent material of solid phase washs with level pad earlier, and then wash with lavation buffer solution, to remove other Yeast Nucleic Acid beyond the messenger RNA(mRNA) and the ribonuclease activity that may exist on the sorbent material, the liquid phase of leaving away is divided in the washing back; The composition of described level pad is: 5～100mmol/L Tutofusin tris-hydrochloric acid of pH7.0～8.0 and 0.5～10mmol/L ethylenediamine tetraacetic acid (EDTA) and 0.4～0.6mol/L sodium-chlor and 0.5～5g/L sodium lauryl sulphate or sodium laurylsulfonate; The composition of described lavation buffer solution is: 5～100mmol/L Tutofusin tris-hydrochloric acid of pH 7.0～8.0 and 0.5～10mmol/L ethylenediamine tetraacetic acid (EDTA) and 0.1～0.2mol/L sodium-chlor and 0.5～5g/L sodium lauryl sulphate or sodium laurylsulfonate;
(5) wash-out: the elution buffer with room temperature～70 ℃ comes out the messenger RNA(mRNA) wash-out that is adsorbed on the sorbent material, collects the elution buffer that contains messenger RNA(mRNA) then; The composition of described elution buffer is: 5～20mmol/L Tutofusin tris-hydrochloric acid of pH7.0～8.0 and 0～5mmol/L ethylenediamine tetraacetic acid (EDTA) and 0～1g/L sodium lauryl sulphate or sodium laurylsulfonate.
2, a kind of method of effectively separating total length, complete messenger RNA(mRNA) from total Yeast Nucleic Acid according to claim 1 is characterized in that:
(1) total Yeast Nucleic Acid is dissolved in the water of the deoxyribonuclease that contains 1g/L sodium lauryl sulphate or sodium laurylsulfonate in the described dissolving step;
(2) total Yeast Nucleic Acid aqueous solution is put into 65 ℃ of water-baths processing 20 minutes in the described denaturing step;
(3) in the described adsorption step:
1. sorbent material adds in the ratio of the total Yeast Nucleic Acid 120mg of 1mg weight in wet base;
2. described affinity chromatography be meant in batches partition method and or column spinner method or column chromatography;
3. described adsorption-buffering liquid is formed: the 20mmol/L Tutofusin tris-hydrochloric acid of pH 7.5 and 2mmol/L ethylenediamine tetraacetic acid (EDTA) and 1mol/L sodium-chlor and 1g/L sodium lauryl sulphate or sodium laurylsulfonate;
4. described sorbent material is oligomerization deoxythymidine-Mierocrystalline cellulose, or the oligomerization deoxythymidine is connected on the polystyrene micelle, or oligomerization uridylic acid-dextrane gel;
5. described absorption is adopted under the room temperature with 30～60 minutes level of 50～80r/min level vibration vibration absorption;
(4) in the described washing step, described level pad adds in the ratio of the total Yeast Nucleic Acid 0.5～1mL of 1mg, and its composition is: the 10mmol/L Tutofusin tris-hydrochloric acid of pH 7.5 and 1mmol/L ethylenediamine tetraacetic acid (EDTA) and 0.5mol/L sodium-chlor and 1g/L sodium lauryl sulphate or sodium laurylsulfonate; Described lavation buffer solution adds in the ratio of the total Yeast Nucleic Acid 0.5～1mL of 1mg, and its composition is: the 10mmol/L Tutofusin tris-hydrochloric acid of pH 7.5 and 1mmol/L ethylenediamine tetraacetic acid (EDTA) and 0.15mol/L sodium-chlor and 1g/L sodium lauryl sulphate or sodium laurylsulfonate;
(5) in the described elution step, the messenger RNA(mRNA) wash-out that is adsorbed on the sorbent material is come out with 65 ℃ elution buffers; Described elution buffer is formed: the 10mmol/L Tutofusin tris-hydrochloric acid of pH7.5 and 1mmol/L ethylenediamine tetraacetic acid (EDTA).
3, a kind of method of effectively separating total length, complete messenger RNA(mRNA) from total Yeast Nucleic Acid according to claim 1 is characterized in that:
(1) described affinity chromatography be in batches partition method and or column spinner method or column chromatography;
(2) described sorbent material is oligomerization deoxythymidine-Mierocrystalline cellulose, or the oligomerization deoxythymidine is connected on the polystyrene micelle, or oligomerization uridylic acid-dextrane gel.
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