CN105802959A - Plant mRNA (messenger ribonucleic acid) extraction method - Google Patents
Plant mRNA (messenger ribonucleic acid) extraction method Download PDFInfo
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- CN105802959A CN105802959A CN201610382799.4A CN201610382799A CN105802959A CN 105802959 A CN105802959 A CN 105802959A CN 201610382799 A CN201610382799 A CN 201610382799A CN 105802959 A CN105802959 A CN 105802959A
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- acupuncture needle
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The invention discloses a plant mRNA (messenger ribonucleic acid) extraction method, belongs to the technical field of molecular biology, and particularly relates to the technical field of extracting mRNA from plant materials through acupuncture needles.The plant mRNA extraction method is provided to solve the problems of operation complexity, low efficiency and damage to plant materials in conventional mRNA extraction methods.The plant mRNA extraction method includes 1, treating an acupuncture needle; 2, putting the dried acupuncture needle into a mixed solution of trimethoxysilane, xylene and diisopropylethylamine for incubation; 3, embedding the acupuncture needle to enable poly-thymine to be adsorbed to the surface of the acupuncture needle; 4, autoclaving the embedded acupuncture needle subjected to ultrapure water elution; 5, punching the naturally dried acupuncture needle into a test position of a target gene of a tender plant material, keeping the acupuncture needle at the test position for 1-3 minutes, pulling out the acupuncture needle, and putting the acupuncture needle into a PCR (polymerase chain reaction) tube of diethyl pyrocarbonate water to complete plant mRNA extraction.The plant mRNA extraction method has the advantages that total RNA extraction is not needed, the mRNA can be extracted from the plant materials in 1-3 minutes, and simplicity, convenience and rapidness of the operation process are achieved.
Description
Technical field
The invention belongs to technical field of molecular biology, be specifically related to extract from vegetable material with acupuncture needle the technical field of mRNA.
Background technology
High-quality RNA is by the important prerequisite of molecular biology of plants research, due to the complicated variety of constituent in plant tissue cell so that the extracting method of plant tissue RNA has multiformity.What commonly use in current research is traditional method, including guanidine isothiocyanate method, phenol-SDS method, CTAB method, Trizol method etc., it all extracts plant total serum IgE under the premise of cell rupture, and test required mRNA and only account for the 1%～4% of total serum IgE, and it is loaded down with trivial details to extract process, efficiency low cost is high.
Summary of the invention
In order to solve traditional method for extracting mRNA complicated operation, efficiency is low, and the problem of infringement vegetable material, it is provided that plant mRNA extracting method.
The plant mRNA extracting method of the present invention sequentially includes the following steps:
One, the process of acupuncture needle: put into by acupuncture needle in brown bottle, in ultrasonic washing unit, successively with hexane, acetone and alcoholic solution vibrations cleaning needle acupuncture needle；Again brown bottle is placed in 75 DEG C～82 DEG C baking ovens dry；
Two, dried acupuncture needle is put in trimethoxy silane, dimethylbenzene and diisopropylethylamine mixed solution and hatch, under 75 DEG C～82 DEG C conditions, cultivate 10h～16h；Operating process use nitrogen isolate oxygen, it is prevented that medicine aoxidizes；The volume ratio of described diisopropylethylamine, dimethylbenzene and trimethoxy silane is 1:(70～80): (20～30)；
Three, the acupuncture needle after cultivating with the ethyl acetate solution cleaning of 48 DEG C～52 DEG C 3～5 times, then acupuncture needle is put in the mixed solution of 0.1 Μ KOH and 0.1-0.5 μ g/ml poly thymus pyrimidine (OligodT), under 35 DEG C～40 DEG C conditions, embed 4h～8h, make poly thymus pyrimidine (OligodT) be adsorbed on acupuncture needle surface；The volume ratio of described KOH and poly thymus pyrimidine is (95～100): 1；
Four, with the acupuncture needle after the embedding of autoclaving ultra-pure water eluting, the acupuncture needle after eluting is put natural drying in atmosphere；
The genes of interest detection position that five, acupuncture needle after natural drying penetrates young tender vegetable material keeps 1min～3min, extracts acupuncture needle and puts in the PCR pipe equipped with 8uL～12uL pyrocarbonic acid diethyl ester (DEPC) water, namely completes plant mRNA and extract.
The present invention is relative to the advantage of prior art:
The invention provides a kind of solid phase mRNA gene and extract new technique, be acupuncture needle be carried out, hatch, cultivate, embed, after dry sequence of operations processes, make OligodT be adsorbed on the needle surface of acupuncture needle, prick, with the acupuncture needle after processing, the position that vegetable material sets, extract its mRNA.Realize the extraction to purpose mRNA.This technology need not extract its total serum IgE, the process processing acupuncture needle is simple, and once can process substantial amounts of acupuncture needle for testing, its process extracting RNA is quick, genes of interest is capable of positioning accurately extraction by the acupuncture needle after process, to specimen material fanout free region or damage, and can extracting mRNA in 1min～3min from vegetable material, operating process is easy, quick.Gene technology transformation at RNA quantitatively, be distributed, transcribe, gene expression research in space-time unique has huge potentiality, there is the significant superiority of functional gene extractive technique than ever, practical extensive with application, China's ornamental plant transgenic breeding, germplasm innovation can be played positive impetus by its use.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the acupuncture needle that embodiment 1 uses.
Detailed description of the invention
Technical solution of the present invention is not limited to act detailed description of the invention set forth below, also includes the combination in any between each detailed description of the invention.
Detailed description of the invention one: the plant mRNA extracting method of present embodiment sequentially includes the following steps:
One, the process of acupuncture needle: put into by acupuncture needle in brown bottle, in ultrasonic washing unit, successively with hexane, acetone and alcoholic solution vibrations cleaning needle acupuncture needle；Again brown bottle is placed in 75 DEG C～82 DEG C baking ovens dry；
Two, dried acupuncture needle is put in trimethoxy silane, dimethylbenzene and diisopropylethylamine mixed solution and hatch, under 75 DEG C～82 DEG C conditions, cultivate 10h～16h；Operating process use nitrogen isolate oxygen, it is prevented that medicine aoxidizes；The volume ratio of described diisopropylethylamine, dimethylbenzene and trimethoxy silane is 1:(70～80): (20～30)；
Three, the acupuncture needle after cultivating with the ethyl acetate solution cleaning of 48 DEG C～52 DEG C 3～5 times, then acupuncture needle is put in the mixed solution of 0.1 Μ KOH and 0.1-0.5 μ g/ml poly thymus pyrimidine (OligodT), under 35 DEG C～40 DEG C conditions, embed 4h～8h, make poly thymus pyrimidine (OligodT) be adsorbed on acupuncture needle surface；The volume ratio of described KOH and poly thymus pyrimidine is (95～100): 1；
Four, with the acupuncture needle after the embedding of autoclaving ultra-pure water eluting, the acupuncture needle after eluting is put natural drying in atmosphere；
The genes of interest detection position that five, acupuncture needle after natural drying penetrates young tender vegetable material keeps 1min～3min, extracts acupuncture needle and puts in the PCR pipe equipped with 8uL～12uL pyrocarbonic acid diethyl ester (DEPC) water, namely completes plant mRNA and extract.
Detailed description of the invention two: present embodiment and detailed description of the invention one are 0.2h～2h the difference is that, step one shakes the time of cleaning needle acupuncture needle.Other steps and parameter are identical with detailed description of the invention one.
Detailed description of the invention three: present embodiment and detailed description of the invention one the difference is that, step one drying time is 0.2h～2h.Other steps and parameter are identical with detailed description of the invention one.
Detailed description of the invention four: present embodiment and detailed description of the invention one the difference is that, trimethoxy silane, dimethylbenzene and diisopropylethylamine mixed solution described in step 2 are the mixed liquor mixing 5s～10s on vortex mixer.Other steps and parameter are identical with detailed description of the invention one.
Detailed description of the invention five: present embodiment and detailed description of the invention one the difference is that, the volume ratio of described diisopropylethylamine, dimethylbenzene and trimethoxy silane is 1:75:25.Other steps and parameter are identical with detailed description of the invention one.
Detailed description of the invention six: present embodiment and detailed description of the invention one are 99:1 the difference is that the volume ratio of, described KOH with poly thymus pyrimidine.Other steps and parameter are identical with detailed description of the invention one.
Embodiment 1
Being put in 1.5mL brown bottle by 10 Φ 0.16 × 7mm acupuncture needles, down, needle handle is down for syringe needle, add hexane solution 1mL, brown bottle is inserted on foam, float over after the ultrasonic washing unit water surface shaking cleaning 15 minutes, taking out, hexane sucking-off thrown away, surplus acupuncture needle is inside；Acetone, alcoholic solution is used to operate cleaning equally more successively；Put in the drying baker being preheating to 80 DEG C in advance by opening the brown bottle of the lid acupuncture needle together with the inside, take out and natural cooling after 15 minutes；Draw 0.25mL trimethoxy silane with rifle and put in 1.5mL centrifuge tube, sequentially add and add 0.75mL dimethylbenzene, the diisopropylethylamine of 10 μ L, mix 5 seconds on vortex mixer afterwards, mixed solution is joined equipped with in the brown bottle of acupuncture needle, cover lid (addition trimethoxy silane, dimethylbenzene, the operation of diisopropyl second are both needed to use nitrogen to isolate oxygen, it is prevented that medicine aoxidizes)；Brown bottle is put in 80 DEG C of drying bakers and cultivate 10 hours；Taking out so as to be reduced to room temperature afterwards, sucking-off mixed liquor discards, and then with acetic acid cleaning needle acupuncture needle, cleans 3 times, uses 1mL every time；Prepare 0.1MKOH solution 99uL, add the 100umOligodT of 1uL, take out wherein 20uL and join in PCR pipe, take dried acupuncture needle 2 simultaneously and add in this PCR pipe；Will be equipped with the PCR pipe of acupuncture needle and be inserted on foam (making to expose bottom PCR pipe), put into together in the water-bath being preheating to 37 DEG C in advance and embed 6 hours；Clean the acupuncture needle that embedded with 50 DEG C of autoclaving water 3 times, clean 5 minutes every time；Acupuncture needle after cleaning is placed in air natural drying；Dried acupuncture needle is penetrated in plant leaf blade and keeps 2 minutes, extract acupuncture needle and put in the PCR pipe equipped with 10uLDEPC water, this PCR pipe it is inserted on foam and is together put in the water-bath being preheating to 80 DEG C in advance, taking out after 10 minutes, namely extract plant mRNA.
Achieving the extraction to purpose mRNA by the present embodiment, and can extract mRNA in 2 minutes from vegetable material, operating process is easy, quick.
Claims (6)
1. plant mRNA extracting method, it is characterised in that: the method sequentially includes the following steps:
One, the process of acupuncture needle: put into by acupuncture needle in brown bottle, in ultrasonic washing unit, successively with hexane, acetone and alcoholic solution vibrations cleaning needle acupuncture needle；Again brown bottle is placed in 75 DEG C～82 DEG C baking ovens dry；
Two, dried acupuncture needle is put in trimethoxy silane, dimethylbenzene and diisopropylethylamine mixed solution and hatch, under 75 DEG C～82 DEG C conditions, cultivate 10h～16h；The volume ratio of described diisopropylethylamine, dimethylbenzene and trimethoxy silane is 1:(70～80): (20～30)；
Three, the acupuncture needle after cultivating with the ethyl acetate solution cleaning of 48 DEG C～52 DEG C 3～5 times, then acupuncture needle is put in the mixed solution of 0.1 Μ KOH and 0.1-0.5 μ g/ml poly thymus pyrimidine, under 35 DEG C～40 DEG C conditions, embed 4h～8h, make poly thymus pyrimidine be adsorbed on acupuncture needle surface；The volume ratio of described KOH and poly thymus pyrimidine is (95～100): 1；
Four, with the acupuncture needle after the embedding of autoclaving ultra-pure water eluting, the acupuncture needle after eluting is put natural drying in atmosphere；
The genes of interest detection position that five, acupuncture needle after natural drying penetrates young tender vegetable material keeps 1min～3min, extracts acupuncture needle and puts in the PCR pipe equipped with 8uL～12uL pyrocarbonic acid diethyl ester water, namely completes plant mRNA and extract.
2. plant mRNA extracting method according to claim 1, it is characterised in that: it is 0.2h～2h that step one shakes the time of cleaning needle acupuncture needle.
3. plant mRNA extracting method according to claim 1, it is characterised in that: step one drying time is 0.2h～2h.
4. plant mRNA extracting method according to claim 1, it is characterised in that: trimethoxy silane, dimethylbenzene and diisopropylethylamine mixed solution described in step 2 are the mixed liquor mixing 5s～10s on vortex mixer.
5. plant mRNA extracting method according to claim 1, it is characterised in that: the volume ratio of described diisopropylethylamine, dimethylbenzene and trimethoxy silane is 1:75:25.
6. plant mRNA extracting method according to claim 1, it is characterised in that: the volume ratio of described KOH and poly thymus pyrimidine is 99:1.
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| CN201610382799.4A CN105802959A (en) | 2016-06-01 | 2016-06-01 | Plant mRNA (messenger ribonucleic acid) extraction method |
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| CN201610382799.4A CN105802959A (en) | 2016-06-01 | 2016-06-01 | Plant mRNA (messenger ribonucleic acid) extraction method |
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Citations (5)
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| CN101165183A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for separating mRNA by using gold magnetism particles |
| CN102154505A (en) * | 2011-04-20 | 2011-08-17 | 苟德明 | Method and primers for detecting mi ribonucleic acid (miRNA) and application of method |
| CN102191326A (en) * | 2011-04-18 | 2011-09-21 | 南京工业大学 | Nucleic acid hybrid film strip for detecting exogenous gene and use thereof |
| CN102978105A (en) * | 2012-12-24 | 2013-03-20 | 天津启仁医药科技有限公司 | mRNA (messenger ribonucleic acid) quick-extraction kit |
| CN103160498A (en) * | 2013-04-08 | 2013-06-19 | 哈尔滨体育学院 | Reverse transcription method of blood mRNA (Messenger Ribose Nucleic Acid) of excellent ice and snow athletes |
-
2016
- 2016-06-01 CN CN201610382799.4A patent/CN105802959A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101165183A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for separating mRNA by using gold magnetism particles |
| CN102191326A (en) * | 2011-04-18 | 2011-09-21 | 南京工业大学 | Nucleic acid hybrid film strip for detecting exogenous gene and use thereof |
| CN102154505A (en) * | 2011-04-20 | 2011-08-17 | 苟德明 | Method and primers for detecting mi ribonucleic acid (miRNA) and application of method |
| CN102978105A (en) * | 2012-12-24 | 2013-03-20 | 天津启仁医药科技有限公司 | mRNA (messenger ribonucleic acid) quick-extraction kit |
| CN103160498A (en) * | 2013-04-08 | 2013-06-19 | 哈尔滨体育学院 | Reverse transcription method of blood mRNA (Messenger Ribose Nucleic Acid) of excellent ice and snow athletes |
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| JAGANNATH B.LAMTURE等: "Direct detection of nucleic acid hybridization on the surface of a charge coupled device", 《OXFORD UNIVERSITY PRESS》 * |
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