CN111647595A - Method for extracting total RNA of winter-resistant camellia leaves - Google Patents
Method for extracting total RNA of winter-resistant camellia leaves Download PDFInfo
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- CN111647595A CN111647595A CN202010582864.4A CN202010582864A CN111647595A CN 111647595 A CN111647595 A CN 111647595A CN 202010582864 A CN202010582864 A CN 202010582864A CN 111647595 A CN111647595 A CN 111647595A
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- 238000000034 method Methods 0.000 title claims abstract description 11
- 235000018597 common camellia Nutrition 0.000 title claims abstract description 10
- 240000001548 Camellia japonica Species 0.000 title abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000002156 mixing Methods 0.000 claims abstract description 10
- 238000001179 sorption measurement Methods 0.000 claims abstract description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000000843 powder Substances 0.000 claims abstract description 8
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- 239000002699 waste material Substances 0.000 claims abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 7
- 239000011259 mixed solution Substances 0.000 claims abstract description 6
- 239000006166 lysate Substances 0.000 claims abstract description 5
- 230000001804 emulsifying effect Effects 0.000 claims abstract description 4
- 238000000227 grinding Methods 0.000 claims abstract description 4
- 239000004570 mortar (masonry) Substances 0.000 claims abstract description 4
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims abstract description 4
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims abstract description 4
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims abstract description 4
- 230000009089 cytolysis Effects 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 4
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 241000209507 Camellia Species 0.000 claims 3
- 238000012163 sequencing technique Methods 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 238000010008 shearing Methods 0.000 abstract 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 14
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000013824 polyphenols Nutrition 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- -1 polysaccharide polyphenol Chemical class 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
Classifications
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- 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
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention aims to provide a method for extracting total RNA of winter camellia leaves, which comprises the steps of shearing tender leaves after tanning from winter camellia plants, adding isovolumic cross-linked polyvinylpyrrolidone and sample powder into a mortar filled with liquid nitrogen for grinding, extracting lysate supernatant, adding isovolumic TRIZOL reagent, adding one fifth of chloroform, uniformly mixing and emulsifying to be milky, standing and centrifuging; extracting supernatant into a new RNase-free tube, adding equal volume of ethanol, and rapidly mixing; adding the mixed solution into an RNA adsorption column, centrifuging, and pouring off waste liquid in the collecting pipe; adding ethanol, centrifuging, and removing waste liquid; placing the RNA adsorption column in a centrifuge tube, adding RNAfree-H2And O, standing at room temperature and centrifuging. The invention has the beneficial effect that the extracted total RNA can be used for full-length transcriptome sequencing or full-transcriptome sequencing analysis on a pacbio platform. Meanwhile, the technology has lower cost than the kit.
Description
Technical Field
The invention belongs to the technical field of RNA gene extraction, and relates to a method for extracting total RNA from leaves of winter camellia.
Background
At present, a kit method is generally adopted for extracting total RNA of plants. The kit has high cost, and has the problems of low concentration, poor integrity and the like, for example, the concentration of RNA extracted by the takara and Tiangen polysaccharide polyphenol type kit is low, the integrity is poor, the degradation of RNA extracted by the bioflux kit is serious and the like. Polysaccharide tissues cannot be extracted by a conventional TRIZOL method, polyphenol tissues cannot be extracted by CTAB, and high-quality winter camellia leaf RNA cannot be effectively obtained by the traditional handheld methods such as SDS.
Disclosure of Invention
The invention aims to provide a method for extracting total RNA from leaves of winter camellia, and the method has the beneficial effect that the extracted total RNA can be used for full-length transcriptome sequencing or full-transcriptome sequencing analysis on a pacbio platform. Meanwhile, the technology has lower cost than the kit.
The technical scheme adopted by the invention is carried out according to the following steps:
1. cutting tender leaves of winter-resistant camellia plants, wrapping with tinfoil, and quickly freezing in liquid nitrogen for later use. Adding isovolumetric cross-linked polyvinylpyrrolidone into a mortar, adding liquid nitrogen while quickly grinding, and making the winter-resistant camellia leaves into powder as soon as possible;
2. adding a lysis solution reagent into the leaf powder, uniformly mixing the leaf powder and the lysis solution on a shaker until no obvious large particles exist, standing for lysis, and centrifuging at low temperature;
3. adding TRIZOL reagent with the same volume into the lysate supernatant, and standing at room temperature;
4. adding one fifth volume of chloroform, mixing, emulsifying to obtain milky white, standing, and centrifuging;
5. extracting supernatant into a new RNase-free tube, adding equal volume of ethanol, and rapidly mixing; adding the mixed solution into an RNA adsorption column, centrifuging, and pouring off waste liquid in the collecting pipe;
6. adding ethanol, centrifuging, and removing waste liquid;
7. placing the RNA adsorption column in a centrifuge tube, adding RNAfree-H2O, standing at room temperature, and centrifuging.
Detailed Description
The present invention will be described in detail with reference to the following embodiments.
1. Cutting tender leaves of winter-resistant camellia plants within 10s, wrapping with tinfoil, and quickly freezing in liquid nitrogen for over 1 h. Adding cross-linked polyvinylpyrrolidone (pvpp) with the same volume into a mortar, adding liquid nitrogen while quickly grinding, and making the winter-resistant camellia leaves into powder as soon as possible;
2. adding 50-100mg leaf powder into lysate Fruit-mateTMfor RNA purification (takara) reagent, mix on the shaker until no obvious large particle state, 4 degrees C static lysis for 5min, 12000Xg, centrifuging at low temperature of 4 ℃ for 5-10min (properly prolonging according to the cracking degree);
3. adding TRIZOL reagent with the same volume into the lysate supernatant, and standing at room temperature for 5-10min (the lysis degree can be properly prolonged);
4. adding one fifth volume (200ul) of chloroform, mixing, emulsifying to give milky white, standing for 5min, and centrifuging at 12000Xg at 4 deg.C for 15 min;
5. extracting supernatant (which does not absorb DNA in the middle layer and protein in the lower layer) to new 1.5ml RNase-free tube, adding 75% ethanol with the same volume, and rapidly mixing; adding the mixed solution into an RNA adsorption column (commercially available), centrifuging at 12000Xg for 30s, and pouring off the waste liquid in the collection tube (adding the mixed solution twice when more mixed solution is available);
6. adding 700ul 75% ethanol, centrifuging at 12000Xg for 30s, and discarding the waste liquid (if the ratio of A260/A230 is lower than 0.5, repeating step 6);
7. 12000Xg, centrifuging for 2 min;
8. placing RNA adsorption column in a 1.5ml centrifuge tube of RNase-free, adding 40-50ul RNAfree-H2O (DEPC (diethyl pyrocarbonate)) is treated overnight, sterilized at high temperature, left to stand at room temperature for 1min, and centrifuged at 12000rpm for 1min (the eluent can be added back to the adsorption column to repeat the operation step 8 if the concentration is not ideal).
9. Storing at-80 deg.C or above.
The method can be used for extracting the RNA of the winter-tolerant camellia with the third generation single-molecule real-time sequencing.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not intended to limit the present invention in any way, and all simple modifications, equivalent variations and modifications made to the above embodiments according to the technical spirit of the present invention are within the scope of the present invention.
Claims (1)
1. A method for extracting total RNA of winter-resistant camellia leaves is characterized by comprising the following steps:
1. cutting tender leaves of the winter camellia plants after tannification, wrapping the tender leaves with tinfoil, quickly freezing the tender leaves in liquid nitrogen for later use, adding isovolumetric cross-linked polyvinylpyrrolidone into a mortar, and quickly grinding the tender leaves while adding the liquid nitrogen to enable the leaves of the winter camellia plants to be powdery as soon as possible;
2. adding a lysis solution reagent into the leaf powder, uniformly mixing the leaf powder and the lysis solution on a shaker until no obvious large particles exist, standing for lysis, and centrifuging at low temperature;
3. adding TRIZOL reagent with the same volume into the lysate supernatant, and standing at room temperature;
4. adding one fifth volume of chloroform, mixing, emulsifying to obtain milky white, standing, and centrifuging;
5. extracting supernatant into a new RNase-free tube, adding equal volume of ethanol, and rapidly mixing; adding the mixed solution into an RNA adsorption column, centrifuging, and pouring off waste liquid in the collecting pipe;
6. adding ethanol, centrifuging, and removing waste liquid;
7. placing the RNA adsorption column in a centrifuge tube, adding RNAfree-H2And O, standing at room temperature and centrifuging.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010582864.4A CN111647595A (en) | 2020-06-23 | 2020-06-23 | Method for extracting total RNA of winter-resistant camellia leaves |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010582864.4A CN111647595A (en) | 2020-06-23 | 2020-06-23 | Method for extracting total RNA of winter-resistant camellia leaves |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111647595A true CN111647595A (en) | 2020-09-11 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202010582864.4A Pending CN111647595A (en) | 2020-06-23 | 2020-06-23 | Method for extracting total RNA of winter-resistant camellia leaves |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002057289A1 (en) * | 2001-01-16 | 2002-07-25 | Invitrogen Corporation | Reagent for the isolation of rna |
| CN104017800A (en) * | 2014-06-20 | 2014-09-03 | 益百尚(北京)生物技术有限责任公司 | Whole genome DNA (Deoxyribonucleic Acid) extraction kit for blood and method thereof |
| CN104371997A (en) * | 2014-11-25 | 2015-02-25 | 福建省农业科学院甘蔗研究所 | Improved method for extracting total RNA in special vegetable tender leaves |
| CN104694531A (en) * | 2015-03-20 | 2015-06-10 | 中国科学院昆明植物研究所 | Method for extracting total RNA from tea tissue |
-
2020
- 2020-06-23 CN CN202010582864.4A patent/CN111647595A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002057289A1 (en) * | 2001-01-16 | 2002-07-25 | Invitrogen Corporation | Reagent for the isolation of rna |
| CN104017800A (en) * | 2014-06-20 | 2014-09-03 | 益百尚(北京)生物技术有限责任公司 | Whole genome DNA (Deoxyribonucleic Acid) extraction kit for blood and method thereof |
| CN104371997A (en) * | 2014-11-25 | 2015-02-25 | 福建省农业科学院甘蔗研究所 | Improved method for extracting total RNA in special vegetable tender leaves |
| CN104694531A (en) * | 2015-03-20 | 2015-06-10 | 中国科学院昆明植物研究所 | Method for extracting total RNA from tea tissue |
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Application publication date: 20200911 |
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