CN105543216A - Method for quickly extracting total RNA of plant tissue - Google Patents
Method for quickly extracting total RNA of plant tissue Download PDFInfo
- Publication number
- CN105543216A CN105543216A CN201610116464.8A CN201610116464A CN105543216A CN 105543216 A CN105543216 A CN 105543216A CN 201610116464 A CN201610116464 A CN 201610116464A CN 105543216 A CN105543216 A CN 105543216A
- Authority
- CN
- China
- Prior art keywords
- plant tissue
- total serum
- serum ige
- rna
- supernatant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to the field of molecular biology, in particular to a method for quickly extracting total RNA from plant tissue. The method comprises steps as follows: a mortar is burnt with alcohol and then quickly precooled with liquid nitrogen, cross-linked polyvinyl pyrrolidone and the plant tissue are added to the mortar and fully ground with liquid nitrogen, then a preheated CTAB (cetyltrimethylammonium bromide) extract solution and mercaptoethanol are added for cell disruption, then chloroform extraction is performed, and the total RNA is obtained through quick precipitation with equal volume of high-concentration LiCl. With the adoption of the method, degradation of RNA by RNase as well as interference of phenolic substances and polysaccharides can be effectively controlled, the consumed time is shorter, the cost is lower, the efficiency is high, the purity and integrity of extracted RNA are good, the requirement for high-purity RNA can be met, and the method has enormous application prospect.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of method of rapid extraction plant tissue total serum IgE.
Background technology
The prerequisite of the molecular biology of plants such as the RNA that DNA purity is high from plant tissue, integrity is good carries out gene clone, gene expression regulation research; In the process extracting RNA, not only can be subject to the pollution of RNase, also can be subject to carbohydrate, polyphenol abundant in plant tissue, the interference of the materials such as protein simultaneously, thus cause the RNA impurity of separation many, purity is low, is difficult to the requirement reaching subsequent experimental; The method used time of extracting RNA in current most plants tissue is long, and working efficiency is low, and the commercial reagents boxes such as TRIZOL, although fast, cost is higher, and its quality and effect are also unstable.
Summary of the invention
Be directed to the above-mentioned problems in the prior art, the object of this invention is to provide a kind of method of rapid extraction plant tissue total serum IgE, the RNA purity that the method is extracted is high, and extraction time is short, substantially increases working efficiency.
To achieve these goals, the technical solution used in the present invention is:
A kind of method of rapid extraction plant tissue total serum IgE is provided, comprises the following steps:
1) mortar clean after, carry out calcination with alcohol, then with the rapid precooling of liquid nitrogen, cross-linking polyethylene pyrrolidone and plant tissue be placed in above-mentioned mortar according to the ratio of 1:1 ~ 1:5 (w/w), and in liquid nitrogen grind into powder; The above-mentioned powder getting about 0.1-0.3g is placed in 2ml centrifuge tube, adds 10 μ l beta-mercaptoethanols and 1ml preheating CTAB extracting solution, concussion mixing, and at 55-68 DEG C, water-bath 20-30 minute, is cooled to room temperature;
2) in step 1) described in centrifuge tube in directly add 1ml chloroform, fully after concussion, under normal temperature, the centrifugal 5min of 12000rpm; Get supernatant, add equal-volume chloroform, fully after concussion, 4 DEG C, the centrifugal 10min of 12000rpm, gets supernatant, repeats extracting once according to same condition;
3) get supernatant, add equal-volume 8MLiCl, precipitate 1-2h under normal temperature, the centrifugal 10min of 12000rpm, thoroughly abandons supernatant, retains precipitation;
4) in above-mentioned precipitation, 75% alcohol immersion 5-10min is added, to turn upside down washing, the centrifugal 5min of 8000rpm, thoroughly abandon supernatant, repeated washing 1-2 time, gained is air-dry 10-15min (not having alcohol residue) under being deposited in room temperature, then adds 20-30 μ lDEPC process water dissolution ,-20 DEG C of preservations.
The mass ratio of described cross-linking polyethylene pyrrolidone and plant tissue is 1:1 (w/w).
Described plant tissue is root, stem, leaf, flower, the fruits and seeds of plant.
Described CTAB extract recipe is: 3%CTAB, 2%PVP, 200mMTris-HCl (pH8.0), 50mMEDTA, 1.4MNaCl.
The present invention also uses liquid nitrogen fast precooling after first being burnt with alcohol by mortar, add the cross-linking polyethylene pyrrolidone of specified proportion and plant tissue in mortar, after fully grinding with liquid nitrogen, the CTAB extracting solution and the beta-mercaptoethanol that add preheating again carry out lysis, then through chloroform, total serum IgE is obtained by isopyknic high density LiCl rapid precipitation.The method can effectively control RNase to the interference of the degraded of RNA, aldehydes matter and polysaccharide, obtained highly purified RNA, and the used time is short, cost is low, and efficiency is high, the RNA purity extracted and integrity high, the demand to highly purified RNA can be met, there is huge application prospect.
Accompanying drawing explanation
Fig. 1 is the gel electrophoresis figure adopting the method for the embodiment of the present invention 1 to extract strawberry different tissues total serum IgE;
Fig. 2 uses the PCR gel electrophoresis figure that the total serum IgE of the embodiment of the present invention 1 extraction is template amplification strawberry Actin gene;
Fig. 3 is the gel electrophoresis figure using the method for the embodiment of the present invention 2 to extract blackberry, blueberry, strawberry, oranges and tangerines, Kiwifruit total serum IgE.
Fig. 4 is the gel electrophoresis figure using the method for the embodiment of the present invention 2 to extract Chinese rose, gladiolus and cabbage mustard total serum IgE.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, below in conjunction with the specific embodiment of the invention and corresponding accompanying drawing, technical solution of the present invention is clearly and completely described.
Embodiment 1
Cleaned by mortar, carry out calcination with alcohol, then liquid nitrogen cools rapidly, adds cross-linking polyethylene pyrrolidone and the 0.2g From Strawberry Leaves of 0.2g, grind into powder in liquid nitrogen environment;
Get the powder of the above-mentioned grinding of 0.11g, be positioned in 2ml centrifuge tube, add 10 μ l beta-mercaptoethanols and the preheated CTAB extracting solution of 1ml, concussion mixing, at 65 DEG C, water-bath 20-30 minute, is cooled to room temperature;
1ml chloroform is added again in above-mentioned centrifuge tube, fully after concussion, under normal temperature, the centrifugal 5min of 12000rpm; Get supernatant, add equal-volume chloroform, fully after concussion, 4 DEG C, the centrifugal 10min of 12000rpm; Get supernatant, add equal-volume chloroform, fully after concussion, 4 DEG C, the centrifugal 10min of 12000rpm;
Get supernatant, add equal-volume 8MLiCl, precipitate 1h under normal temperature, the centrifugal 10min of 12000rpm, thoroughly abandons supernatant, is precipitated;
75% alcohol immersion 5min is added, washing of turning upside down, the centrifugal 5min of 8000rpm in above-mentioned precipitation, thoroughly abandon supernatant, according to same condition repeated washing 2 times, gained is air-dry 15min under being deposited in room temperature, add 20ulDEPC process water dissolution again, preserve at-20 DEG C and use.
Embodiment 2
The plant tissue adopted is respectively the stem of strawberry, flower, fruit and seed, and extraction step is with embodiment 1, and the total serum IgE of extraction is respectively stem total serum IgE, flower total serum IgE, fruit total serum IgE and seed total serum IgE.
Embodiment 3
Mortar is cleaned, carries out calcination with alcohol, then cool rapidly with liquid nitrogen, add cross-linking polyethylene pyrrolidone and the 1g blackberry, blueberry fruit of 0.2g, grind into powder in liquid nitrogen environment;
Get the powder of the above-mentioned grinding of 0.23g, be positioned in 2ml centrifuge tube, add 10ul beta-mercaptoethanol and the preheated CTAB extracting solution of 1ml, concussion mixing, at 60 DEG C, water-bath 20-30 minute, is cooled to room temperature;
1ml chloroform is added again in above-mentioned centrifuge tube, fully after concussion, under normal temperature, the centrifugal 5min of 12000rpm; Get supernatant, add after equal-volume chloroform fully shakes, 4 DEG C, the centrifugal 10min of 12000rpm; Get supernatant, add after equal-volume chloroform fully shakes, 4 DEG C, the centrifugal 10min of 12000rpm;
Get supernatant, add equal-volume 8MLiCl, precipitate 2h under normal temperature, the centrifugal 10min of 12000rpm, thoroughly abandons supernatant, is precipitated;
75% alcohol immersion 10min is added, washing of turning upside down, the centrifugal 5min of 8000rpm in above-mentioned precipitation, thoroughly abandon supernatant, according to same condition repeated washing 2 times, gained is air-dry 10min under being deposited in room temperature, add 20ulDEPC water dissolution again, obtained, preserve at-20 DEG C and use.
Embodiment 4
The plant tissue adopted is respectively strawberry fruit, Citrus leaf, Kiwifruit blade, Chinese rose petal, gladiolus kind ball and cabbage mustard seed, extraction step is with embodiment 3, and the total serum IgE of extraction is respectively strawberry total serum IgE, oranges and tangerines total serum IgE, Kiwifruit total serum IgE, Flos Rosae Chinensis total serum IgE, gladiolus total serum IgE and cabbage mustard total serum IgE.
The total serum IgE of the strawberry different tissues that Example 1-2 extracts, by its OD value (as shown in table 1 below) of UV spectrophotometer measuring, result shows: the concentration of the total serum IgE of extraction is higher, the ratio of OD260/OD280 is between 1.92-2.08, the ratio of OD260/OD230, between 1.91-2.16, illustrates that the quality of the total serum IgE extracted from the different tissues of different plant is higher.
Carry out electrophoresis detection with 1% sepharose to above-mentioned total serum IgE, as shown in Figure 1, result shows: RNA band is clear, and integrity is better, and the brightness of 28S band is 2 times of 18S, and does not almost have the pollution of DNA; In FIG, 1, root; 2, stem; 3, leaf; 4, flower; 5, fruit; 6, seed.
By the RNA of said extracted after reverse transcription with 1 couple of Actin primer (5'-TGGGTTTGCTGGAGATGA-3'; 5'-CAGTTAGGAGAACTGGGTG-3') carry out pcr amplification, obtain the set goal band, as shown in Figure 2; In fig. 2,1, root; 2, stem; 3, leaf; 4, flower; 5, fruit; 6, seed.
The OD value that table 1 strawberry different tissues RNA extracts detects
The total serum IgE of the different tissues of the different plants that Example 3-4 extracts, by its OD value (as shown in table 2 below) of UV spectrophotometer measuring, result shows: the concentration of the total serum IgE of extraction is higher, the ratio of OD260/OD280 is between 1.90-2.06, the ratio of OD260/OD230, between 1.94-2.15, illustrates that the quality of the total serum IgE extracted from the different tissues of the different plants such as blackberry, blueberry, strawberry, oranges and tangerines, Kiwifruit, Chinese rose, gladiolus and cabbage mustard is higher.
Carry out electrophoresis detection with 1% sepharose to above-mentioned total serum IgE, as shown in Figure 3, Figure 4, result shows: RNA band is clear, and integrity is better, and the brightness of 28S band is 2 times of 18S, and does not almost have the pollution of DNA; In figure 3,1, blackberry, blueberry; 2, strawberry; 3, oranges and tangerines; 4, Kiwifruit; In the diagram, 1, Chinese rose; 2, gladiolus; 3, cabbage mustard.
The OD value that the different plant tissue RNA of table 2 extracts detects
As can be seen from the result of above-mentioned several embodiment, the strawberry different tissues RNA extracted by the inventive method and the RNA of other plant different tissues is purer, DNA and protein-polysaccharide etc. pollute not serious, RNA band is more clear simultaneously, integrity is better, and may be used for follow-up clone and the experiment such as quantitatively.
Claims (4)
1. a method for rapid extraction plant tissue total serum IgE, is characterized in that, comprises the following steps:
1) after mortar is cleaned, with alcohol calcination mortar, then with the rapid precooling of liquid nitrogen, by cross-linking polyethylene pyrrolidone (PVPP) and plant tissue according to 1:1 ~ 1:5(w/w) ratio be placed in above-mentioned mortar, and use liquid nitrogen grinding powdered; The above-mentioned powder getting 0.1-0.3g is placed in 2ml centrifuge tube, adds 10 μ l beta-mercaptoethanols and 1ml preheating CTAB extracting solution, concussion mixing, and at 55-68 DEG C, water-bath 20-30 minute, is cooled to room temperature;
2) in the centrifuge tube described in step 1), 1ml chloroform is directly added, fully after concussion, under normal temperature, the centrifugal 5min of 12000rpm; Get supernatant, add equal-volume chloroform, fully after concussion, 4 DEG C, the centrifugal 10min of 12000rpm, gets supernatant, repeats extracting once according to same condition;
3) get supernatant, add equal-volume 8MLiCl, precipitate 1-2h under normal temperature, the centrifugal 10min of 12000rpm, thoroughly abandons supernatant, retains precipitation;
4) in above-mentioned precipitation, 75% alcohol immersion 5-10min is added, washing of turning upside down, the centrifugal 5min of 8000rpm, thoroughly abandon supernatant, repeated washing 1-2 time, gained under being deposited in room temperature air-dry 10-15min to alcohol-free residual, add 20-30 μ lDEPC process water dissolution again ,-20 DEG C of preservations.
2. the method for rapid extraction plant tissue total serum IgE according to claim 1, is characterized in that, the mass ratio of described cross-linking polyethylene pyrrolidone and plant tissue is 1:1(w/w).
3. the method for rapid extraction plant tissue total serum IgE according to claim 1, is characterized in that, described plant tissue is root, stem, leaf, flower, the fruits and seeds of plant.
4. the method for rapid extraction plant tissue total serum IgE according to claim 1, is characterized in that, described CTAB extract recipe is:
3%CTAB，2%PVP，200mMTris-HCl(pH8.0)，50mMEDTA，1.4MNaCl。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610116464.8A CN105543216A (en) | 2016-03-02 | 2016-03-02 | Method for quickly extracting total RNA of plant tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610116464.8A CN105543216A (en) | 2016-03-02 | 2016-03-02 | Method for quickly extracting total RNA of plant tissue |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105543216A true CN105543216A (en) | 2016-05-04 |
Family
ID=55822774
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610116464.8A Pending CN105543216A (en) | 2016-03-02 | 2016-03-02 | Method for quickly extracting total RNA of plant tissue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105543216A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107603975A (en) * | 2017-11-13 | 2018-01-19 | 山西农业大学 | A kind of lily method for extracting total RNA |
| CN108277218A (en) * | 2018-03-31 | 2018-07-13 | 中国农业科学院草原研究所 | A kind of Leymus chinensis seeds RNA extraction method at sprouting initial stage |
| CN110195055A (en) * | 2019-07-02 | 2019-09-03 | 南宁维尔凯生物科技有限公司 | Polysaccharide polyphenol plant tissue method for extracting total RNA |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050191739A1 (en) * | 1995-05-23 | 2005-09-01 | Commonwealth Scientific And Industrial Research Organisation | Polyphenol oxidase genes from banana, lettuce, tobacco and pineapple |
| CN101649355A (en) * | 2009-09-02 | 2010-02-17 | 北京林业大学 | Method for detecting lily seed virus |
| CN101948819A (en) * | 2010-08-26 | 2011-01-19 | 中国农业大学 | Oxyneurin metabolic plant tyrosine phosphatase protein, coding gene thereof and use thereof |
| CN102061297A (en) * | 2010-08-04 | 2011-05-18 | 陕西师范大学 | Transgenic method for improving salvianolic acid B content in root of red-rooted salvia |
| CN102220353A (en) * | 2011-04-28 | 2011-10-19 | 西南大学 | Encoding sequence of Cinnamic acid-4-hydroxylase protein in sweet potato and application of same |
| CN102586235A (en) * | 2012-01-13 | 2012-07-18 | 浙江省农业科学院花卉研究开发中心 | Method for extracting total ribonucleic acid (RNA) from bracts and inflorescences of Anthurium andraeanum |
| CN103232997A (en) * | 2013-04-18 | 2013-08-07 | 中国热带农业科学院分析测试中心 | Method for extracting total RNA from leucaena leucocephala leaf tissue |
-
2016
- 2016-03-02 CN CN201610116464.8A patent/CN105543216A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050191739A1 (en) * | 1995-05-23 | 2005-09-01 | Commonwealth Scientific And Industrial Research Organisation | Polyphenol oxidase genes from banana, lettuce, tobacco and pineapple |
| CN101649355A (en) * | 2009-09-02 | 2010-02-17 | 北京林业大学 | Method for detecting lily seed virus |
| CN102061297A (en) * | 2010-08-04 | 2011-05-18 | 陕西师范大学 | Transgenic method for improving salvianolic acid B content in root of red-rooted salvia |
| CN101948819A (en) * | 2010-08-26 | 2011-01-19 | 中国农业大学 | Oxyneurin metabolic plant tyrosine phosphatase protein, coding gene thereof and use thereof |
| CN102220353A (en) * | 2011-04-28 | 2011-10-19 | 西南大学 | Encoding sequence of Cinnamic acid-4-hydroxylase protein in sweet potato and application of same |
| CN102586235A (en) * | 2012-01-13 | 2012-07-18 | 浙江省农业科学院花卉研究开发中心 | Method for extracting total ribonucleic acid (RNA) from bracts and inflorescences of Anthurium andraeanum |
| CN103232997A (en) * | 2013-04-18 | 2013-08-07 | 中国热带农业科学院分析测试中心 | Method for extracting total RNA from leucaena leucocephala leaf tissue |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107603975A (en) * | 2017-11-13 | 2018-01-19 | 山西农业大学 | A kind of lily method for extracting total RNA |
| CN108277218A (en) * | 2018-03-31 | 2018-07-13 | 中国农业科学院草原研究所 | A kind of Leymus chinensis seeds RNA extraction method at sprouting initial stage |
| CN110195055A (en) * | 2019-07-02 | 2019-09-03 | 南宁维尔凯生物科技有限公司 | Polysaccharide polyphenol plant tissue method for extracting total RNA |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104194389B (en) | A kind of method extracting capsanthin and capsaicine with fresh capsicum processing | |
| CN101638651B (en) | Method for extracting total RNA from plant tissue rich in polysaccharides and polyphenols and secondary metabolites | |
| CN105543216A (en) | Method for quickly extracting total RNA of plant tissue | |
| Rouholamin et al. | Expression analysis of anthocyanin biosynthesis key regulatory genes involved in pomegranate (Punica granatum L.) | |
| CN108410863B (en) | Efficient extraction method of guava leaf genome DNA | |
| CN105949163A (en) | Method for extracting and purifying anthocyanin in pomace of aronia melanocarpa | |
| CA2890482A1 (en) | A method for preparing bioactive botanical compositions and the compositions made from said method | |
| CN101864414A (en) | Extraction method of apostichopus japonicus body-wall total RNA | |
| CN103272008A (en) | Technology for synergistically extracting peony flavone | |
| CN103819517B (en) | Preparation method of tea saponin | |
| CN101899435A (en) | High-throughput extraction method for sugarcane leaf genome by using ball mill | |
| CN104292294A (en) | Sapindus saponin purification method | |
| CN107541506B (en) | Method for extracting DNA of wild allied species of Xinjiang garlic | |
| CN108165663B (en) | Method for identifying sex of date palm tree | |
| Ahmed et al. | Extraction of high-quality intact DNA from okra leaves despite their high | |
| CN115413682B (en) | Application of tomato stem and leaf extract in preventing and treating thrips and composition | |
| CN105524036A (en) | High-efficiency low-consumption grape seed procyanidine extraction method | |
| CN106801051B (en) | Kit for extracting plant RNA and extraction method | |
| Bošeľová et al. | Comparative analysis of different methods of Hedera helix DNA extraction and molecular evidence of the functionality in PCR | |
| Guan et al. | Identification of superior clones by RAPD technology in Xanthoceras sorbifolia Bge. | |
| CN105943589A (en) | Extracting method of snakegourd fruit extract for treating coronary heart disease | |
| CN101928473A (en) | Method for producing lycopene oleoresin | |
| CN112774245A (en) | A method for extracting and separating active extract of semen Canarii albi by supercritical technology | |
| Razik et al. | Analysis of oil palm clones, their suspension calli and regenerants via flow cytometry (FCM) and rDNA-fluorescence in situ hybridisation (rDNA-FISH) | |
| CN101974512B (en) | Tomato total RNA extraction method suitable for microRNAs analysis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160504 |
|
| RJ01 | Rejection of invention patent application after publication |