CN103834637A - Method for extracting nucleic acid by using magnetic nanoparticles and application thereof - Google Patents
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Abstract
The invention provides a method for extracting nucleic acid by using magnetic nanoparticles and an application thereof. The method comprises the following steps: I, cracking a sample by using a cracking buffer liquid to obtain a sample cracking liquid; II, adding 5-100 microliters of liquid of magnetic nanoparticles into 20-100 microliters of the obtained sampling cracking liquid, adding 20-500 microliters of combined buffer liquid, uniformly mixing, transferring into a centrifugal tube, keeping on a magnetic frame for 10-50 seconds, and removing the liquid after nucleic acid is adsorbed and separated by the magnetic nanoparticles to obtain magnetic nanoparticles adsorbed with the nucleic acid; III, adding 50-200 microliters of washing buffer liquid into the magnetic nanoparticles adsorbed with the nucleic acid, uniformly mixing, keeping on the magnetic frame for 10-50 seconds, washing at least once, and removing the liquid; and IV, adding 20-50 microliters of elution buffer liquid into the washed magnetic nanoparticles adsorbed with the nucleic acid, uniformly mixing, keeping on the magnetic frame for 10-50 seconds, and collecting the eluent after fully eluting to obtain the nucleic acid is in the eluent. By adopting the method, the extraction of miRNA (micro Ribonucleic Acid) is realized for the first time, and high extraction efficiency is achieved.
Description
Technical field
The present invention relates to extracting method and the application of a kind of magnetic nanoparticle to nucleic acid, belong to technical field of molecular biology.
Background technology
Contemporary Biology, medical researches etc. have been deep into molecular level, and such as nucleic acid aspect, nucleic acid extraction isolation technique seems particularly important efficiently.Such as the research of examination miRNA tumor markers in the body fluid such as serum/plasma, just need a kind of simple miRNA extractive technique efficiently.
The most conventional technology is classical phenol/chloroform extraction method at present, utilizes the principle of extraction, isolates nucleic acid.But these organic solvents of phenol/chloroform have huge murder by poisoning to human body, also unfavorable on the impact of environment, and operating process complexity, and result is affected by human factors larger, are difficult for realizing automated operation.
Also have in addition some commercial companies that the test kit of the extraction based on centrifugal column method is provided, sample is crossed to post, utilize nucleic acid different with the adsorptive power of pillar under different condition, reach the effect of separation.Operate also more complicated, yield is on the low side, is difficult for realizing automated operation.
Also there are some business-like paramagnetic particle methods to extract test kit, based on the superhigh specific surface area of nanometer magnetic bead, utilize the physical propertys such as electrostatic attraction to realize the separation and purification of nucleic acid, but the leaching process of these test kits or more complicated, step is various, and not yet having the product extracting for miRNA specially to occur, the extraction effect of existing test kit is also difficult to satisfactory.
Summary of the invention
In view of the defect that above-mentioned prior art exists, the object of the invention is to propose extracting method and the application of a kind of magnetic nanoparticle to nucleic acid, can extract miRNA, can improve the extraction efficiency of nucleic acid, can simplify the operation and be beneficial to realize automatization extraction.
Object of the present invention is achieved by the following technical programs:
The extracting method of magnetic nanoparticle to nucleic acid, comprises the steps:
Step 1, is used lysis buffer lysate sample, obtains sample dissociation liquid, and wherein, the proportionlity of the consumption of lysis buffer and the consumption of sample is the lysis buffer correspondence 10 of every 40 μ L
6the sample of cell quantity;
Step 2, the solution of the magnetic nanoparticle of 5 μ L-100 μ L is joined in the sample dissociation liquid of 20 μ L-100 μ L gained, add again the binding buffer liquid of 20 μ L-500 μ L, mix, be transferred in centrifuge tube, be placed on magnetic frame magnetic resolution 10 seconds-50 seconds, after magnetic nanoparticle fractionation by adsorption nucleic acid, remove liquid, obtained adsorbing the magnetic nanoparticle of nucleic acid;
Step 3, to having adsorbed the lavation buffer solution that adds 50 μ L-200 μ L in the magnetic nanoparticle of nucleic acid, mixes, and is placed on magnetic frame magnetic resolution 10 seconds-50 seconds, after washing at least one times, removes liquid;
Step 4, to washing absorption later in the magnetic nanoparticle of nucleic acid, add the elution buffer of 20 μ L-50 μ L, mix, be placed on magnetic frame magnetic resolution 10 seconds-50 seconds, after abundant wash-out, collect elutriant, nucleic acid is in gained elutriant.
In above-mentioned extracting method, sample comprises cell, tissue, paraffin section, and the sample free for nucleic acid do not need cracking, such as serum, blood plasma and urine, directly carries out the operation of step 2.For cell sample, need to first collect culturing cell, centrifugal, remove supernatant liquor, and then add lysis buffer to carry out the operation of step 1.Need to first add xylene soluble for paraffin section, the then centrifugal dimethylbenzene that removes, the aqueous solution cleans twice operation that adds again lysis buffer to carry out step 1 repeatedly.
Above-mentioned magnetic nanoparticle is in the extracting method of nucleic acid, preferably, described lysis buffer comprises following component: sodium lauryl sulphate (SDS) 0.05wt%-0.5wt%, guanidinium isothiocyanate (GITC) 0.1M-4M, proteolytic enzyme k 10 μ g/mL-800 μ g/mL, EDTA 1mmol/L-10mmol/L, NaCl 10mmol/L-150mmol/L.
Above-mentioned magnetic nanoparticle is in the extracting method of nucleic acid, and preferred, described lysis buffer comprises following component: sodium lauryl sulphate 0.05wt%, guanidinium isothiocyanate 0.5M, proteolytic enzyme k 800 μ g/mL, EDTA 10mmol/L, NaCl 150mmol/L.
Above-mentioned magnetic nanoparticle is in the extracting method of nucleic acid, and preferred, described binding buffer liquid comprises following component: NaCl 0.5M-5M, and guanidinium isothiocyanate 0.01M-4M, it is 2.2-6 that Lin acid hydrogen Er Na – citrate buffer solution is adjusted binding buffer liquid pH.
Above-mentioned magnetic nanoparticle is in the extracting method of nucleic acid, and preferred, described binding buffer liquid comprises following component: NaCl 3M, and guanidinium isothiocyanate 0.05M, the pH that Lin acid hydrogen Er Na – citrate buffer solution is adjusted binding buffer liquid is 4.
Above-mentioned magnetic nanoparticle is in the extracting method of nucleic acid, and preferred, described lavation buffer solution comprises following component: NaCl 0.5M-5M, and guanidinium isothiocyanate 0.05M-0.5 M, ethanol 10v%-70v%, it is 2.2-6 that phosphoric acid buffer is adjusted lavation buffer solution pH.
Above-mentioned magnetic nanoparticle is in the extracting method of nucleic acid, and preferred, described lavation buffer solution comprises following component: NaCl 1M, and guanidinium isothiocyanate 0.05M, ethanol 20v%, it is 5 that phosphoric acid buffer is adjusted lavation buffer solution pH.
Above-mentioned magnetic nanoparticle is in the extracting method of nucleic acid, preferred, the Tris-hydrochloride buffer that described elution buffer is pH8.0-10.0.Preferably, the Tris-hydrochloride buffer that described elution buffer is pH10.0.
Above-mentioned magnetic nanoparticle is in the extracting method of nucleic acid, and preferred, described magnetic nanoparticle is nanometer magnetic bead, and its diameter is 100nm-30 μ m, and its finishing group comprises carboxyl, amino.
In above-mentioned extracting method, the magnetic nanoparticle of employing (being nanometer magnetic bead) is core-shell type Magnetic Polystyrene Microsphere, finishing carboxyl, amino or other group.But the magnetic bead of other types also can substitute use.Its principle is: the group that carry on nanometer magnetic bead surface is under the condition of binding buffer liquid, and surface shows positive charge, the nucleic acid of adsorption zone negative charge; Under the condition of elutriant, the aobvious negative charge in surface, and separate nucleic acid.
The present invention also provides the application of the extracting method of a kind of the above magnetic nanoparticle to nucleic acid, for extracting miRNA, or for extracting RNA or DNA.
Technical scheme of the present invention is that to have paramagnetic nanometer magnetic bead be that carrier is (such as the polystyrene core shell-type magnetic bead of surperficial carboxyl modified is under low pH condition, surface band positive charge), directly with sample to be extracted, such as: body fluid (serum, blood plasma, urine etc.) or cell pyrolysis liquid etc., interact.Wherein electronegative nucleic acid forms mixture by electrostatic interaction and nano particle, under the effect of outside magnetic field, realizes separating of mixture and sample to be extracted.Under the effect of elutriant, magnetic bead surfaces electric charge transfers negative charge to afterwards, and the mutual exclusion of nucleic acid electric charge, and nucleic acid is dissolved in elutriant, then under the effect of outside magnetic field, realizes separating of magnetic bead and nucleic acid.The nucleic acid solution finally obtaining can be for the experiment in downstream, such as RT-PCR etc.
Outstanding effect of the present invention is: realize first the extraction to miRNA, also can extract RNA and DNA; Environmental protection, is not used the poisonous chemical reagent such as phenol chloroform; Extraction efficiency is high, especially for small segment nucleic acid such as miRNA; Simple and fast, without complex apparatus; Being applicable to automatization extracts.
Accompanying drawing explanation
Fig. 1 is the schema of the extracting method of magnetic nanoparticle of the present invention to nucleic acid;
Fig. 2 is the RT-qPCR figure (amplification curve diagram) of the gradient extraction of external source cel-mir-39 in embodiment 2;
Fig. 3 is the efficiency comparison diagram that embodiment 6 the inventive method and RNeasy Plus Mini Kit extract the RNA of gene U6 in cell.
Embodiment
Below just accompanying drawing in conjunction with the embodiments, is described in further detail the specific embodiment of the present invention, so that technical solution of the present invention is easier to understand, grasp, but the present invention is not limited thereto.Experimental technique described in following embodiment, if no special instructions, is ordinary method; Described reagent and material, if no special instructions, all can obtain from commercial channels.
Embodiment 1
The present embodiment provides the extracting method of a kind of magnetic nanoparticle to nucleic acid, as shown in Figure 1 (serum sample does not need to add lysis buffer), for the extraction of the endogenous miRNA of serum, comprises the steps:
Step 1, get 100 μ L serum samples, add 100 μ L (to comprise component: NaCl 3M in conjunction with liquid damping fluid, guanidinium isothiocyanate 0.05M, the pH that phosphorus acid hydrogen two sodium – citrate buffer solutions are adjusted binding buffer liquid is 4), the nanometer magnetic bead (the polystyrene core shell-type magnetic bead of surperficial carboxyl modified) of 40 μ L solid contents 0.5%, vibration mixes rear placement 10 minutes, is placed in magnetic resolution on magnetic frame, leave standstill 15 seconds, remove liquid;
Step 2, adds 100 μ L washings damping fluids (to comprise component: NaCl 1M, guanidinium isothiocyanate 0.05M, ethanol 20v%, it is 5 that phosphoric acid buffer is adjusted lavation buffer solution pH), vibration mixes and is placed on magnetic resolution on magnetic frame, leave standstill 15 seconds, remove liquid, repeat once;
Step 3, adds the elution buffer (the Tris-hydrochloride buffer of pH10.0) of 30 μ L, and vibration mixes and is placed on magnetic resolution on magnetic frame, leaves standstill 15 seconds, collects elutriant, and nucleic acid is in gained elutriant.
Get serum sample A, B, C, D, E, the F of 6 Different Individual, operation as above, is extracted product RT-qPCR method and is detected miRNA.
Specific experiment data are as shown in table 1, the measured value that table 1 is the miRNA that extracts in each serum sample.
Table 1
Shown by above-mentioned experimental result, present method can extract the miRNA of trace in serum.
Embodiment 2
The present embodiment provides the extracting method of a kind of magnetic nanoparticle to nucleic acid, and take the outer miRNAs of serum as standard substance, extraction efficiency is detected.
Preparation cel-mir-39 standard solution, three concentration gradients are as follows:
Solution a:cel-mir-39 100pM
Solution b:cel-mir-39 10pM
Solution c:cel-mir-39 1pM.
Get respectively the solution a of 30 μ L, b, c sneaks in the human serum of 100 μ L separately, according to the step operation of embodiment 1, obtains the extraction product A of 30 μ L, B, C.6 duplicate samples are carried out identical RT-qPCR operation simultaneously, and repetitive operation once, the data that obtain are as shown in Fig. 2 (curve a in figure: triangular marker, b: cross mark, c: rectangle marked, A: circles mark, B: diamond indicia, C: unmarked) and table 2.
Table 2
| ? | Ct value |
| Sample | cel-mir-39 |
| a | 18.56 |
| a | 19.07 |
| A | 20.22 |
| A | 19.44 |
| b | 22.17 |
| b | 22.79 |
| B | 22.33 |
| B | 22.92 |
| c | 26.18 |
| c | 26.19 |
| C | 26.66 |
| C | 26.41 |
Wherein, the data of sample A and sample a approach, and the data of sample B and sample b approach, and the data of sample C and sample c approach.The data obtained shows, for the miRNA of different concns, the extraction efficiency of present method all approaches ideal value, and namely present method can almost entirely be extracted miRNA in serum, and magnetic nanoparticle of the present invention has higher extraction efficiency to the extracting method of nucleic acid in serum.
Embodiment 3
The present embodiment provides the extracting method of a kind of magnetic nanoparticle to nucleic acid, and as shown in Figure 1, for the extraction of culturing cell endogenous nucleic acid, step is as follows:
Step 1, collect culturing cell (7 powers of <10), centrifugal, remove supernatant liquor, add lysis buffer and (comprise component: sodium lauryl sulphate 0.05wt%, guanidinium isothiocyanate 0.5M, proteolytic enzyme k 800 μ g/mL, EDTA 10mmol/L, NaCl 150mmol/L) 50 μ L, after lysis, add 400 μ L water dilutions;
Step 2, add again 50 μ L (to comprise component: NaCl 3M in conjunction with liquid, guanidinium isothiocyanate 0.05M, the pH that Lin acid hydrogen Er Na – citrate buffer solution is adjusted binding buffer liquid is 4), the nanometer magnetic bead of 50 μ L solid contents 0.5%, vibration mixes rear placement 10 minutes, is placed in magnetic resolution on magnetic frame, leave standstill 15 seconds, remove liquid;
Step 3, adds 100 μ L lavation buffer solutions (comprise component: NaCl 1M, guanidinium isothiocyanate 0.05M, ethanol 20v%, it is 5 that phosphoric acid buffer is adjusted lavation buffer solution pH), and vibration mixes and is placed on magnetic resolution on magnetic frame, leaves standstill 15 seconds, removes liquid.Repeat once;
Step 4, adds the elution buffer (the Tris-hydrochloride buffer of pH10.0) of 30 μ L, and vibration mixes and is placed on magnetic resolution on magnetic frame, leaves standstill 15 seconds, collects elutriant, and nucleic acid is in gained elutriant.
Get culturing cell NC, be divided into four parts, be numbered 99,192,196, NC, add respectively the agonist of miRNA, 99 add hsa-mir-99b-5p, and 192 add hsa-mir-192b-5p, and 196 add hsa-mir-196b-5p, and NC is without interpolation.Cultivate after for some time, after extracting, detect with RT-qPCR according to as above step, data are as shown in table 3.
Table 3
| Sample | Gene | Ct value | Average Ct value | Ct value | Ct value |
| 99 | hsa-mir-99b-5p | 28.95 | ? | ? | ? |
| 99 | hsa-mir-99b-5p | 28.9 | ? | ? | ? |
| 99 | hsa-mir-99b-5p | 28.9 | 28.9166666666667 | 14.2566666666667 | -7.67666666666666 |
| 99 | U6 | 14.37 | ? | ? | ? |
| 99 | U6 | 14.57 | ? | ? | ? |
| 99 | U6 | 15.04 | 14.66 | ? | ? |
| 192 | hsa-mir-192-5p | 18.81 | ? | ? | ? |
| 192 | hsa-mir-192-5p | 18.81 | ? | ? | ? |
| 192 | hsa-mir-192-5p | 18.27 | 18.63 | 2.2 | -13.35 |
| 192 | U6 | 16.58 | ? | ? | ? |
| 192 | U6 | 16.22 | ? | ? | ? |
| 192 | U6 | 16.49 | 16.43 | ? | ? |
| 196 | hsa-mir-196b-5p | 16.95 | ? | ? | ? |
| 196 | hsa-mir-196b-5p | 17.34 | ? | ? | ? |
| 196 | hsa-mir-196b-5p | 17.17 | 17.1533333333333 | -0.243333333333332 | -12.7033333333333 |
| 196 | U6 | 17.44 | ? | ? | ? |
| 196 | U6 | 17.51 | ? | ? | ? |
| 196 | U6 | 17.24 | 17.3966666666667 | ? | ? |
| ? | ? | ? | ? | ? | ? |
| NC | hsa-mir-99b-5p | 39.89 | ? | ? | ? |
| NC | hsa-mir-99b-5p | 40.17 | ? | ? | ? |
| NC | hsa-mir-99b-5p | 40.29 | 40.1166666666667 | 21.9333333333333 | ? |
| NC | hsa-mir-192-5p | 33.7 | ? | ? | ? |
| NC | hsa-mir-192-5p | 33.9 | ? | ? | ? |
| NC | hsa-mir-192-5p | 33.6 | 33.7333333333333 | 15.55 | ? |
| NC | hsa-mir-196b-5p | 30.6 | ? | ? | ? |
| NC | hsa-mir-196b-5p | 31.19 | ? | ? | ? |
| NC | hsa-mir-196b-5p | 30.14 | 30.6433333333333 | 12.46 | ? |
| NC | U6 | 17.96 | ? | ? | ? |
| NC | U6 | 18.51 | ? | ? | ? |
| NC | U6 | 18.08 | 18.1833333333333 | ? | ? |
Above-mentioned experimental result shows, can well detect the miRNA content in cell, and as can be seen here, the inventive method can be extracted miRNAs in cell preferably.
Embodiment 4
The present embodiment provides the extracting method of a kind of magnetic nanoparticle to nucleic acid, as shown in Figure 1, and for the extraction of culturing cell exogenous nucleic acid.
Step 1, collect culturing cell (7 powers of <10), centrifugal, remove supernatant liquor, add lysis buffer and (comprise component: sodium lauryl sulphate 0.05wt%, guanidinium isothiocyanate 0.5M, proteolytic enzyme k 800 μ g/mL, EDTA 10mmol/L, NaCl 150mmol/L) 50 μ L, after lysis, add 400 μ L water dilutions;
Step 2, adds 50 μ L cel-mir-39 solution (100pM) to step 1 in the split product of the 450 μ L that obtain, obtain mixed solution, then moves into respectively 15 μ L in other 3 empty pipe, 60 μ L, and the mixed solution of 240 μ L, each self-watering is supplied 450 μ L;
Step 3, add 50 μ L (to comprise component: NaCl 3M in conjunction with liquid to three pipes again, guanidinium isothiocyanate 0.05M, the pH that Lin acid hydrogen Er Na – citrate buffer solution is adjusted binding buffer liquid is 4), the nanometer magnetic bead of 50 μ L solid contents 0.5%, vibration mixes rear placement 10 minutes, is placed in magnetic resolution on magnetic frame, leave standstill 15 seconds, remove liquid;
Step 4, adds 100 μ L lavation buffer solutions (comprise component: NaCl 1M, guanidinium isothiocyanate 0.05M, ethanol 20v%, it is 5 that phosphoric acid buffer is adjusted lavation buffer solution pH), and vibration mixes and is placed on magnetic resolution on magnetic frame, leaves standstill 15 seconds, removes liquid.Repeat once;
Step 5, adds the elution buffer (the Tris-hydrochloride buffer of pH10.0) of 30 μ L, and vibration mixes and is placed on magnetic resolution on magnetic frame, leave standstill 15 seconds, collect elutriant, nucleic acid in gained elutriant, obtain the each 30 μ L of corresponding extraction product solution C 1, C2, C3.
Configure in addition contrast solution D1, D2, D3 solution is as follows:
The solution cel-mir-39 solution (100pM) of solution D 1:1.5 μ L, adds the water of 28.5 μ L;
The solution cel-mir-39 solution (100pM) of solution D 2:6 μ L, adds the water of 24 μ L;
The solution cel-mir-39 solution (100pM) of solution D 3:24 μ L, adds the water of 6 μ L.
Repeat once to test simultaneously and carry out, detect the Ct value of the cel-mir-39 in C1, C2, C3, D1, D2, D3 with RT-qPCR, data are as shown in table 4 below.
Table 4
| ? | Ct value |
| Sample | cel-mir-39 |
| C1 | 23.92 |
| C1 | 23.92 |
| C2 | 27.32 |
| C2 | 27.22 |
| C3 | 31.29 |
| C3 | 31.43 |
| D1 | 23.52 |
| D1 | 24.33 |
| D2 | 29.74 |
| D2 | 31.32 |
| D3 | 32.54 |
| D3 | 34.17 |
In table, the data of sample D1 and sample C1 approach, and the data of sample D2 and sample C2 approach, and the data of sample D3 and sample C3 approach.Result shows that the miRNA efficiency that present method is extracted in cell approaches ideal value, and namely present method can almost entirely be extracted extracellular miRNAs, and magnetic nanoparticle of the present invention has higher extraction efficiency to the extracting method of cell exogenous nucleic acid.
Embodiment 5
By magnetic nanoparticle of the present invention, the extracting method to nucleic acid and Trizol method compare the present embodiment.
The present embodiment method is with the method for embodiment 2, take cel-mir-39 as standard substance.Comparative result is as shown in table 5.
Table 5
| ? | Ct value |
| Sample | cel-mir-39 |
| Trizol method | 35.62 |
| Trizol method | 35.98 |
| Paramagnetic particle method | 26.32 |
| Paramagnetic particle method | 26.22 |
From experimental data chart, the Ct value of Trizol method is greater than the inventive method, and namely Trizol method is not so good as present method to the extraction efficiency of miRNA.
Embodiment 6
The present embodiment provides the extracting method of a kind of magnetic nanoparticle to nucleic acid and the comparison of Qiagen test kit (RNeasy Plus Mini Kit).Extracting method (the inventive method) concrete steps of magnetic nanoparticle to nucleic acid are with embodiment 3, the cell of two groups of same quantity, extract with the inventive method and Qiagen test kit respectively, product carries out RT-qPCR and detects U6 content, (the numerical curve triangular marker of the inventive method as shown in Figure 3, the numerical curve circles mark of RNease Plus mini Kit method), illustrate that the efficiency of the inventive method extraction nucleic acid is suitable with Qiagen test kit.
Embodiment 7
The present embodiment provides the extracting method of a kind of magnetic nanoparticle to DNA in culturing cell.Concrete steps, with embodiment 3, are extracted the DNA content of the direct qPCR detection of product gene U6.D40 group cell quantity is 4 times of D10 group, and result is as shown in table 6, and result demonstration the inventive method can well be extracted the DNA in cell.
Table 6
| ? | Ct value |
| Sample | U6 |
| D10 | 27.66 |
| D10 | 27.63 |
| D40 | 26.38 |
| D40 | 26.31 |
Therefore the inventive method has realized the extraction to miRNA first, also can extract RNA and DNA; Environmental protection; Extraction efficiency is high, especially for small segment nucleic acid such as miRNA; Simple and fast, without complex apparatus; Being applicable to automatization extracts.
Claims (10)
1. the extracting method of magnetic nanoparticle to nucleic acid, comprises the steps:
Step 1, is used lysis buffer lysate sample, obtains sample dissociation liquid, and wherein, the proportionlity of the consumption of lysis buffer and the consumption of sample is the lysis buffer correspondence 10 of every 40 μ L
6the sample of cell quantity;
Step 2, the solution of the magnetic nanoparticle of 5 μ L-100 μ L is joined in the sample dissociation liquid of 20 μ L-100 μ L gained, add again the binding buffer liquid of 20 μ L-500 μ L, mix, be transferred in centrifuge tube, be placed on magnetic frame magnetic resolution 10 seconds-50 seconds, after magnetic nanoparticle fractionation by adsorption nucleic acid, remove liquid, obtained adsorbing the magnetic nanoparticle of nucleic acid;
Step 3, to having adsorbed the lavation buffer solution that adds 50 μ L-200 μ L in the magnetic nanoparticle of nucleic acid, mixes, and is placed on magnetic frame magnetic resolution 10 seconds-50 seconds, after washing at least one times, removes liquid;
Step 4, to washing absorption later in the magnetic nanoparticle of nucleic acid, add the elution buffer of 20 μ L-50 μ L, mix, be placed on magnetic frame magnetic resolution 10 seconds-50 seconds, after abundant wash-out, collect elutriant, nucleic acid is in gained elutriant.
2. the extracting method of magnetic nanoparticle according to claim 1 to nucleic acid, it is characterized in that: described lysis buffer comprises following component: sodium lauryl sulphate 0.05wt%-0.5wt%, guanidinium isothiocyanate 0.1M-4M, proteolytic enzyme k 10 μ g/mL-800 μ g/mL, EDTA 1mmol/L-10mmol/L, NaCl 10mmol/L-150mmol/L.
3. the extracting method of magnetic nanoparticle according to claim 2 to nucleic acid, it is characterized in that: described lysis buffer comprises following component: sodium lauryl sulphate 0.05wt% guanidinium isothiocyanate 0.5M, proteolytic enzyme k 800 μ g/mL, EDTA 10mmol/L, NaCl 150mmol/L.
4. the extracting method of magnetic nanoparticle according to claim 1 to nucleic acid, it is characterized in that: described binding buffer liquid comprises following component: NaCl 0.5M-5M, guanidinium isothiocyanate 0.01M-4M, it is 2.2-6 that Lin acid hydrogen Er Na – citrate buffer solution is adjusted binding buffer liquid pH.
5. the extracting method of magnetic nanoparticle according to claim 4 to nucleic acid, it is characterized in that: described binding buffer liquid comprises following component: NaCl 3M, guanidinium isothiocyanate 0.05M, the pH that Lin acid hydrogen Er Na – citrate buffer solution is adjusted binding buffer liquid is 4.
6. the extracting method of magnetic nanoparticle according to claim 1 to nucleic acid, it is characterized in that: described lavation buffer solution comprises following component: NaCl 0.5M-5M, guanidinium isothiocyanate 0.05M-0.5 M, ethanol 10v%-70v%, it is 2.2-6 that phosphoric acid buffer is adjusted lavation buffer solution pH.
7. the extracting method of magnetic nanoparticle according to claim 6 to nucleic acid, is characterized in that: described lavation buffer solution comprises following component: NaCl 1M, and guanidinium isothiocyanate 0.05M, ethanol 20v%, it is 5 that phosphoric acid buffer is adjusted lavation buffer solution pH.
8. the extracting method of magnetic nanoparticle according to claim 1 to nucleic acid, is characterized in that: the Tris-hydrochloride buffer that described elution buffer is pH8.0-10.0, and preferred, the Tris-hydrochloride buffer that described elution buffer is pH10.0.
9. the extracting method of magnetic nanoparticle according to claim 1 to nucleic acid, is characterized in that: described magnetic nanoparticle is nanometer magnetic bead, and its diameter is 100nm-30 μ m, and its finishing group comprises carboxyl, amino.
10. an application for the extracting method of the magnetic nanoparticle described in claim 1-9 any one to nucleic acid, for extracting miRNA, or for extracting RNA or DNA.
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