CN102250887A - Method for extracting RNA (ribonucleic acid) from articular cartilage - Google Patents
Method for extracting RNA (ribonucleic acid) from articular cartilage Download PDFInfo
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- CN102250887A CN102250887A CN 201110225851 CN201110225851A CN102250887A CN 102250887 A CN102250887 A CN 102250887A CN 201110225851 CN201110225851 CN 201110225851 CN 201110225851 A CN201110225851 A CN 201110225851A CN 102250887 A CN102250887 A CN 102250887A
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Abstract
The invention relates to an improved method for extracting RNA (ribonucleic acid) from articular cartilage, and the method is characterized by comprising the following steps: freezing the cartilage tissue at -20 DEG C or lower, and pulverizing with a pulverizer into powder; adding a composite denaturing liquid to the pulverized cartilage to obtain a homogenate, and centrifuging and collecting supernatant; adding polyethylene glycol octyl phenyl ether protein denaturant until the concentration of the denaturant is 0.25-0.35% (V/V), cooling in an ice bath, and extracting the supernatant multiple times with phenol-chloroform-isoamyl alcohol mixed solvent; adding ammonium acetate or sodium acetate until the final concentration is 0.3 M, and then precipitating RNA with isopropanol or ethanol; and further purifying the precipitate with a RNA purification kit. The RNA obtained from articular cartilage in the method provided by the invention has high yield and high purity; the quality and quantity of the obtained RNA are sufficient for the research of the gene expression in chondrocytes; the RNA yield increases by 5-8 times compared with that in conventional methods; the ratio of 260 mu m UV (ultraviolet) absorption value to 280 mu m UV absorption value of the RNA is not less than 2; the original characteristics of the RNA does not change; and by the method, the two major problems that the RNA extracted from cartilage cells by the prior art has low yield and low purity are solved.
Description
Technical field
The present invention relates to a kind of high quality rna method of from tissue, extracting, especially from joint cartilage, extract the method for high quality rna expeditiously, with usefulness for further analysis, detection.
Background technology
The damage of sacroiliitis cartilage has a strong impact on human body health, can cause the intensive arthralgia, even can cause the joints lose mobility.For seeking repairing articular cartilage damage effective means, prior art has generation, the evolution of the Protocols in Molecular Biology research articular cartilage damage of application of advanced.And the changes in gene expression of understanding the damage process chondrocyte in depth is one of very important link, and the RNA (Yeast Nucleic Acid) that extracts sufficient amount and quality is the prerequisite that the work of chondrocyte's gene expression research is carried out for analysis.Yet (Chomczynski P, Sacchi N were published in Anal Biochem. 1987 Apr to RNA guanidine thiocyanate extracting method conventional, that be widely used in various animal tissuess in 1987; 162 (1): 156-9.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.), it is to organize directly homogenate in guanidine thiocyanate solution, uses phenol-chloroform mixed-solvent extraction then, ethanol sedimentation.Actual being difficult to of this method extracted enough high-purity RNA effectively from articular cartilage tissue, gained RNA yield is very low, and purity is not high, makes RNA is analyzed, detects, studies and bring difficulty.Its major cause is: contain a large amount of moisture and extracellular matrix protein in the cartilaginous tissue, the chondrocyte only accounts for about 3% of whole tissue weight, and the chondrocyte is wrapped up by a large amount of cartilage matrixs again, cell is difficult for separating from matrix, like this in conventional guanidine thiocyanate method is extracted a considerable amount of chondrocytes still by in the matrix parcel, fail from matrix, to separate, cause the RNA yield very low thus; Matrix glycoprotein and RNA can form co-precipitation in addition, make RNA be difficult to be further purified.It is low that these factors have caused conventional guanidine thiocyanate method to extract in the joint cartilage RNA yield and purity, directly influenced follow-up research work.
Chinese patent CN101182343 method for extracting total RNA of animal articular cartilage tissue, (1) gets animal articular cartilage tissue, drop into immediately be ground in the liquid nitrogen Powdered, the articular cartilage tissue of per 50 weight parts adds 1 volume sex change liquid (guanidinium isothiocyanate 23.6320 weight parts, trisodium citrate 0.3676 weight part, sarcosyl 0.25 weight part, add 25 weight part tri-distilled waters, be settled to 50 volumes), the beta-mercaptoethanol of 0.007 volume, homogenate is to there not being obvious tissue block; (2) homogenate is placed on the ice-water bath, add acid water-saturated phenol and shake up, add 2mol/L concentration sodium-acetate, chloroform, primary isoamyl alcohol mixed solution (chloroform: primary isoamyl alcohol is 49: 1) place 15min on the ice-water bath; Under (3) 4 ℃, centrifugal, get supernatant liquor, add chloroform, primary isoamyl alcohol mixed solution, the centrifuging and taking supernatant liquor adds Virahol, and-20 ℃ precipitate 30min, centrifugal 30 minutes down, obtain gel RNA and protein-polysaccharide mixed precipitation, add no RNA enzyme water, dissolution precipitation obtains colloidal solution; (4) get colloidal solution and obtain total RNA precipitation, get precipitation, precipitate with no RNA enzyme water dissolution behind the washing with alcohol secondary and promptly get the articular cartilage tissue total rna solution with the RNA extraction step of plant RNAout test kit.The applicant tests discovery, since cartilaginous tissue in liquid nitrogen freezing after, very hard, the powder particle that forms by grinding is bigger, homogenate is to there not being obvious tissue block, also showing has fine particle to exist, and causes still having a considerable amount of chondrocytes to fail to be released, and can cause final RNA yield low relatively; Secondly, adopt " obtaining gel RNA and protein-polysaccharide mixed precipitation; add no RNA enzyme water; dissolution precipitation obtains colloidal solution ", can make RNA and a large amount of protein-polysaccharide form co-precipitation, the albumen that causes removing to mix wherein is difficult relatively, and making often has albumen to leave in final RNA sample, and this can form the interference to follow-up study work.
Above-mentioned deficiency still has is worth improved place.
Summary of the invention
The object of the invention is to overcome above-mentioned the deficiencies in the prior art, and a kind of yield height is provided, the highly purified method of extracting RNA from joint cartilage.
The object of the invention realizes that mainly improving one is with the cartilaginous tissue cryogenic freezing, with the pulverizer pulverizing, to improve the yield of isolation of chondrocytes; The 2nd, the ice bath cooling is extracted behind the adding Triton X-100, with raising isolation of RNA purity, thereby overcomes the deficiencies in the prior art, realizes the object of the invention.Specifically, the present invention extracts the method for RNA from joint cartilage, it is characterized in that with cartilaginous tissue-20 ℃ and following freezing be ground into pulverizer Powdered; In the cartilaginous tissue of pulverizing, add complex denaturation liquid and make homogenate, centrifugal collection supernatant liquor; Add the Triton X-100 protein denaturant, making its concentration is 0.25-0.35% V/V, and repeatedly extracting supernatant liquor of phenol-chloroform-primary isoamyl alcohol mixed solvent is used in the ice bath cooling; Add ammonium acetate or sodium acetate, then with Virahol or ethanol sedimentation RNA; Throw out is further purified with RNA purifying medicine box.
Before the detailed description, make a presentation earlier, so that those skilled in the art have one clearly to understand to this patent general plotting technical scheme and the basic effect that reaches by the basic function and the effect that can reach to invention.
The inventive method, cartilaginous tissue cryogenic freezing, pulverizer are pulverized, make full use of cartilaginous tissue and under freezing, can become very hard crisp, test is found to pulverize with pulverizer, can overcome grinding and have the particulate deficiency so that cartilaginous tissue is easy to fully be pulverized, made the chondrocyte be able to from matrix, at utmost discharge, help improving later separation RNA yield, pulverize at low temperatures simultaneously and produce the little RNA that also easily guarantees of heat relatively not by sex change; Select to adopt Triton X-100 (commercial disignation Triton X-100) protein denaturant and in ice bath, cool off, help further fully sex change of various albumen in the crushed material, make RNA can separate more fully with matrix glycoprotein etc., help albumen and in follow-up phenol-chloroform-primary isoamyl alcohol mixed-solvent extraction process, fully removed, thereby improved the purity of obtaining RNA.
Among the present invention.
In-20 ℃ and following environment, pulverize, just can more fully cartilaginous tissue fully be pulverized, test finds that the low more crushing effect of freezing temp is good more, therefore a kind of deep hypothermia freezing (for example-80 ℃ and following) that more preferably adopts is pulverized for example freezing in being full of the pulverizer of liquid nitrogen, abundant pulverizing.Used freezing crusher, the Spex Freezer Mill pulverizer of Spex Industries company product is more better adopted in test, can obtain the fine powder of enough fineness, makes the chondrocyte be able at utmost discharge from matrix.
Adding ammonium acetate or sodium acetate, mainly is that RNA is precipitated under the salinity environment.It is 0.3-0.5 M (mol) that test better adds ammonium acetate or sodium acetate to ultimate density, RNA is precipitated fully, if salt concn is low excessively in higher salt concentrations, then precipitation can be not exclusively, salt concn is too high, salt can with the RNA coprecipitation, can influence with post analysis.
RNA purifying medicine box, its effect are to be further purified cartilage RNA sample solution, remove the albumen that still may leave over, and reduced volume, finally obtain the high quality rna sample.Though RNA purifying medicine box can both be used in the prior art, compare the RNeasy RNA purifying medicine box that QIAGEN company (U.S.) produces through test, have the better purification effect of RNA purifying medicine box usually, thereby preferentially be used.
In addition, for the more abundant protein denaturation that makes is easy to remove, and the integrity of assurance RNA, test complex denaturation liquid better adopts by 0.1-0.15 M Tutofusin tris, pH7.0-7.5; 20-25 mM ethylenediamine tetraacetic acid (EDTA), pH 7.0-7.5; 0.5-0.6 microlitre/milliliter mercaptoethanol; 3.5-4.5 the compound guanidine thiocyanate solution that the M guanidine thiocyanate is formed.
In the inventive method, do not mention method, the step of explanation especially, to extract the RNA method from animal tissues identical with prior art, can directly be employed.
The present invention extracts the method for RNA from joint cartilage, with respect to prior art, owing to adopt freezing, pulverizer pulverizing, make that cartilaginous tissue is easy to fully be pulverized, overcome the deficiency of prior art employing freeze grinding, make the chondrocyte be able to from matrix, at utmost discharge, thereby improved the RNA yield; Adding Triton X-100 protein denaturant also cools off in ice bath, help the further fully sex change of various albumen, make RNA can separate more completely with matrix glycoprotein etc., help albumen and in follow-up phenol-chloroform-primary isoamyl alcohol mixed-solvent extraction process, fully removed, thereby improved the purity of obtaining RNA.Adopt the compound guanidine thiocyanate system handles among the present invention, make protein denaturation more abundant thereby be easier to remove, and help better guaranteeing the integrity of RNA.The inventive method is for the effective means that provides of high quality rna is provided from cartilaginous tissue efficiently, experiment shows the RNA that adopts the inventive method to obtain, has the yield height, the purity height, its quality and quantity all is enough to be used for carrying out the research of chondrocyte's genetic expression, thereby provide the foundation for further studying chondrocyte's expression of gene, solved prior art and from the chondrocyte, extracted the low and low two large problems of purity of RNA yield.Detection shows: the RNA that adopts the inventive method to obtain, not only its yield is much higher than conventional extraction means, improve 5-8 doubly than ordinary method, and the RNA that extracts has very high purity, ratio 〉=2 of 260 μ m ultraviolet absorption values/280 μ m ultraviolet absorption values of RNA (showing that purity is very high), and any change does not take place in the primary characteristic of gained RNA.Because the RNA of its hetero-organization of animal is relatively than the easier degraded of cartilaginous tissue, thereby extracts than easier in the cartilaginous tissue, so the inventive method not only can be used for extracting RNA from cartilaginous tissue, and the RNA that obviously more can be used for its hetero-organization of animal extracts.
Below in conjunction with a specific embodiment; essence of the present invention is further understood in exemplary illustration and help; but the embodiment detail only is for the present invention is described; do not represent the present invention to conceive whole technical schemes down; therefore should not be construed as the technical scheme qualification total to the present invention, some are In the view of the technician, and the unsubstantiality that does not depart from the present invention's design increases and/or change; for example simple the change or replacement of technical characterictic to have same or similar technique effect all belongs to protection domain of the present invention.
Description of drawings
Fig. 1 is RNA blotting test gel electrophoresis result.Among the figure
Fibronectin mRNA pattern among A animal (horse) the liver organization RNA
Fibronectin mRNA pattern among B animal (horse) the cartilaginous tissue RNA
Fibronectin mRNA pattern among C animal (horse) the cartilaginous tissue RNA
Fibronectin mRNA pattern among D animal (horse) the muscle tissue RNA
E animal (horse) is cultivated fibronectin mRNA pattern among the chondrocyte RNA.
Fig. 2 is polymerase chain reaction (PCR) gel electrophoresis result.Among the figure
AWith horse cartilaginous tissue RNA is the distinctive fibronectin gene-splicing of the cartilaginous tissue pattern that the template chain reaction amplifies
BWith horse liver organization RNA is the fibronectin gene-splicing pattern that the template chain reaction amplifies
CWith horse cartilaginous tissue RNA is the distinctive fibronectin gene-splicing of the cartilaginous tissue pattern that the template chain reaction amplifies
DWith horse muscle tissue RNA is the fibronectin gene-splicing pattern that the template chain reaction amplifies
EWith rabbit cartilaginous tissue RNA is the distinctive fibronectin gene-splicing of the cartilaginous tissue pattern that the template chain reaction amplifies.
Embodiment
Embodiment: gather the cartilaginous tissue sample from the horse knee joint, its tissue sample is stored in-80
ZeroStand-by under the C low temperature.
1, gets about 25 gram cartilaginous tissue samples, place pulverizer (the Spex Freezer Mill) sample hose that is full of liquid nitrogen, at full throttle pulverize 3 times, be not shorter than 1 minute at every turn.
2, get cartilaginous tissue after 1-2 gram is pulverized, add the compound guanidine thiocyanate solution of 30-40 milliliter, add mercaptoethanol to 0.5-0.6 microlitre/milliliter, high-speed homogenization (Bio-Gen PRO200 homogenizer temporarily, 10000-15000 rpm speed homogenate 3 times, in each 10 seconds, cool off 10 seconds, homogenate once more) at ice bath, low-speed centrifugal (1500-2000g, 10-15 minute, 4 ° of C), collect supernatant liquor.
3, in throw out, add the compound guanidine thiocyanate solution of 30-40 milliliter once more, repeat above-mentioned homogenate, centrifugation step.Merge twice supernatant liquor (final volume is about the 45-65 milliliter).
4, adding Triton X-100, to make ultimate density be 0.20-0.25% V/V, mixing; Placed 15-30 minute in the ice bath.Phenol-chloroform-primary isoamyl alcohol (24:24:1) mixed solution that adds equal volume, fully concussion is evenly left standstill, and takes out supernatant liquor; Repeat no longer to exist until white middle layer for several times.
5, adding ammonium acetate or sodium acetate solution to final concentration are 300-500mM (this example is 400mM) in supernatant liquor, place 30-60 minute in ice bath behind the mixing, add the Virahol of 0.8 volume again, place 30-60 minute in ice bath behind the mixing again; High speed centrifugation (15000-20000g, 30-45 minute, 4 ° of C).Abandoning supernatant, throw out be dry air 10 minutes at room temperature.
6, the lysis buffer (RLT) the RNeasy that adds RNA purifying medicine box (QIAGEN(u s company) product in throw out), the step according to the medicine box defined is further purified sample then, and dissolving RNA sample.
Measure the ultraviolet absorption value of gained RNA at 260 μ m and 280 μ m with ultraviolet spectrophotometer: the ratio of 260 μ m absorption values/280 μ m absorption values is 2.2-2.3, illustrates that RNA purity reaches requirement.
Compound guanidine thiocyanate formulations prepared from solutions with 0.1-0.15 M Tutofusin tris, is regulated pH7.0-7.5 with hydrochloric acid; 20-25 mM ethylenediamine tetraacetic acid (EDTA) (EDTA), pH 7.0-7.5; 0.5-0.6 microlitre/milliliter mercaptoethanol (2-Mercaptoethanol); 3.5-4.5 M guanidine thiocyanate (Guanidinium Thiocyanate), the supernatant liquor that carries out homogenate and centrifugal collection; Repeat above step twice, merge all supernatant liquors.
Comparative example: extract cartilaginous tissue RNA with aforementioned conventional Chomczynski method, final gained RNA purity is on the low side, and 260 μ m absorption values/280 μ m absorption values are 1.6-1.8.
Both compare, and the inventive method improves 6 times than the ordinary method yield.
Extract horse liver organization, muscle tissue, cultivation chondrocyte's RNA respectively by example 1 method.
With 5 kinds of RNA that the inventive method is extracted, carry out the test of RNA blotting, result such as Fig. 1 show that by figure the mRNA among 5 kinds of RNA is very complete, the RNA that shows the inventive method extraction is without any qualitative change; 5 kinds of RNA with the inventive method preparation are template again, carry out polymerase chain reaction (PCR); By all not observing the miscellaneous background in each sample of Fig. 2 photo, prove that fully the RNA that extracts has very high purity.By analysis to the polymerase chain reaction product, found a kind of new fibronectin gene distinctive shear mode in cartilaginous tissue, prove the vital role that the high quality rna of present method preparation is risen in the research of follow-up chondrocyte's genetic expression.
To those skilled in the art; under this patent design and specific embodiment enlightenment; some distortion that can directly derive or associate from this patent disclosure and general knowledge; those of ordinary skills will recognize also can adopt additive method; or the substituting of known technology commonly used in the prior art; and the equivalence of feature changes or modification; mutual various combination between feature; for example complex denaturation liquid changes, and for example increases or reduces discrete component in complex liquid, or adopt complex denaturation liquid of the prior art; the change of pulverizer; the change of freezing temp, the change of purification reagent kit kind, or the like unsubstantiality change; can be employed equally; can both realize this patent representation function and effect, launch for example no longer one by one to describe in detail, all belong to this patent protection domain.
Claims (7)
1. from joint cartilage, extract the method for RNA, it is characterized in that with cartilaginous tissue-20 ℃ and following freezing be ground into pulverizer Powdered; In the cartilaginous tissue of pulverizing, add complex denaturation liquid and make homogenate, centrifugal collection supernatant liquor; Add the Triton X-100 protein denaturant, making its concentration is 0.25-0.35% V/V, and repeatedly extracting supernatant liquor of phenol-chloroform-primary isoamyl alcohol mixed solvent is used in the ice bath cooling; Add ammonium acetate or sodium acetate, then with Virahol or ethanol sedimentation RNA; Throw out is further purified with RNA purifying medicine box.
2. according to the described method of from joint cartilage, extracting RNA of claim 1, it is characterized in that freezing in liquid nitrogen, to carry out.
3. according to the described method of extracting RNA from joint cartilage of claim 1, it is characterized in that pulverizing with pulverizer is the Spex Freezer Mill pulverizer that Spex Industries company produces.
4. according to the described method of from joint cartilage, extracting RNA of claim 1, it is characterized in that complex denaturation liquid is by 0.1-0.15 M Tutofusin tris, pH7.0-7.5; 20-25 mM ethylenediamine tetraacetic acid (EDTA), pH 7.0-7.5; 0.5-0.6 microlitre/milliliter mercaptoethanol; 3.5-4.5 the compound guanidine thiocyanate solution that the M guanidine thiocyanate is formed.
5. according to the described method of from joint cartilage, extracting RNA of claim 1, it is characterized in that RNA purifying medicine box is the Rneasy that QIAGEN produces.
6. according to the described method of extracting RNA from joint cartilage of claim 1, it is characterized in that adding ammonium acetate or sodium acetate to ultimate density is 0.3-0.5M.
7. according to claim 1,2,3,4, the 5 or 6 described methods of from joint cartilage, extracting RNA, it is characterized in that ratio 〉=2 of 260 μ m ultraviolet absorption values/280 μ m ultraviolet absorption values of gained RNA.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642797A (en) * | 2013-12-11 | 2014-03-19 | 华中农业大学 | Extraction method of total RNA of intermuscular bone of megalobrama amblycephala |
| CN107699559A (en) * | 2016-08-08 | 2018-02-16 | 山西农业大学 | A kind of method from animal cartilaginous tissue extraction RNA |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182343A (en) * | 2007-12-18 | 2008-05-21 | 中国人民解放军第三军医大学第二附属医院 | Method for extracting total RNA of animal articular cartilage tissue |
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2011
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182343A (en) * | 2007-12-18 | 2008-05-21 | 中国人民解放军第三军医大学第二附属医院 | Method for extracting total RNA of animal articular cartilage tissue |
Non-Patent Citations (2)
| Title |
|---|
| 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 19960802 James N. MacLeod, et al. Fibronectin mRNA Splice Variant in Articular Cartilage Lacks Bases Encoding the V, III-15, and I-10 Protein Segments. 18954-18960 1-7 第271卷, 第31期 * |
| 《中国中药杂志》 20080831 黄月琴等. 半夏块茎高质量总RNA提取的一种有效方法. 1810-1813 1-7 第33卷, 第15期 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642797A (en) * | 2013-12-11 | 2014-03-19 | 华中农业大学 | Extraction method of total RNA of intermuscular bone of megalobrama amblycephala |
| CN107699559A (en) * | 2016-08-08 | 2018-02-16 | 山西农业大学 | A kind of method from animal cartilaginous tissue extraction RNA |
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Application publication date: 20111123 |