CN103740700A - Extracting method for total RNA (Ribonucleic Acid) of galangal - Google Patents
Extracting method for total RNA (Ribonucleic Acid) of galangal Download PDFInfo
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- CN103740700A CN103740700A CN201310749752.3A CN201310749752A CN103740700A CN 103740700 A CN103740700 A CN 103740700A CN 201310749752 A CN201310749752 A CN 201310749752A CN 103740700 A CN103740700 A CN 103740700A
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Abstract
The invention discloses an extracting method for the total RNA (Ribonucleic Acid) of galangal. The method is improved on various aspects on the basis of the conventional guanidine thiocyanate-phenol-chloroform extraction method according to the characteristic of rich secondary metabolism product of the galangal and particularly rich polyhydric flavonol, i.e., acetone, water-soluble polyvinylpyrrolidone (PVP) and beta-mercaptoethanol are added in an RNA extracting solution. The acetone can be used for dissolving colored matters such as flavonols and the like; the water-soluble PVP can be sufficiently combined with phenol substances to form chelates; the beta-mercaptoethanol as a strong reducing agent can be used for providing a reducing condition so that polyphenol substances are not easy to oxidize; the three substances synergistically take effects, so that the interference and obstruction of the polyphenol substances are effectively inhibited. According to the invention, a crudely-extruded sediment of the RNA is treated by using Tris saturated phenol and high-concentration potassium acetate after being obtained, and part of proteins and polysaccharides can be simultaneously removed.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the extracting method of the total RNA of a kind of medicinal plant, particularly the extracting method of the total RNA of a kind of Rhizoma Alpiniae Officinarum.
Background technology
From plant tissue, DNA purity RNA high, that integrity is good is cDNA reverse transcription in the researchs such as plant gene function and expression regulation, In Vitro Translation, cDNA library foundation, prerequisite and the key of RT-PCR and differential analysis etc.In practice, often can find, even if its RNA extracting method of the different tissues of same plant can be very different; Or even same plant same organization material, but deriving from different genotype plant, its RNA extracting method also may be different.So for a certain plant or its tissue, its corresponding RNA extracting method must be through groping and putting into practice and could establish.
Rhizoma Alpiniae Officinarum (Alpinia officinarum Hance), calls little galingal, is one of famous southern medicine of China, has more than 1,000 year medicinal history.Rhizoma Alpiniae Officinarum, except as taste of traditional Chinese medicine side, is also widely used in the preparation of food, makeup and drug additive.China produces Rhizoma Alpiniae Officinarum and has found a good sale in the Middle East and America and Europe.
Rhizoma Alpiniae Officinarum is organized the complicated variety of secondary metabolite, poly-hydroxy flavonols rich content particularly, flavonols is very easily oxidized, its oxidation products can cause RNA degraded and loss of activity or form insoluble mixture, and flavonols of a great variety, content is very high can cause Rhizoma Alpiniae Officinarum to organize RNA to extract difficulty.
Not yet having at present document or patent to relate to Rhizoma Alpiniae Officinarum RNA extracts and follow-up molecular studies.
Summary of the invention
Applicant has attempted the plant RNA extraction method of various kinds of document report, and RNA extraction commercial kit (comprising the test kit that extracts specially development for polysaccharide polyphenol plant tissue RNA) is carried out extracting to Rhizoma Alpiniae Officinarum plant tissue RNA, but result is all undesirable, most methods and test kit can not extract RNA; Only a few extracted RNA, but degraded is serious, can not carry out follow-up study.Therefore, the object of the present invention is to provide the extracting method of the total RNA of a kind of Rhizoma Alpiniae Officinarum, the method is reproducible, can obtain the RNA of high quality, high density.
Object of the present invention is achieved through the following technical solutions:
An extracting method of the total RNA of Rhizoma Alpiniae Officinarum, comprises the following steps:
1) get Rhizoma Alpiniae Officinarum young leaflet tablet and put into precooling mortar, in mortar, add precooling cracking extract and pre-cold acetone, repeatedly add liquid nitrogen grinding to become powder, room temperature is placed 30-40min, gained homogenate is transferred in centrifuge tube, 4 ℃, the centrifugal 15min of 12000g, shift colourless supernatant layer to another centrifuge tube, obtains solution A; Described cracking extract is comprised of following component: 5mol/L guanidinium isothiocyanate, 25mmol/L Trisodium Citrate, 0.5%(w/v) sarcosyl, 4%(w/v) soluble poly V-Pyrol RC (PVP, K40), 4%(v/v) beta-mercaptoethanol;
Described Rhizoma Alpiniae Officinarum blade is liquid nitrogen flash freezer young leaflet tablet or fresh young leaflet tablet;
2) in solution A, add the saturated phenol of Tris (pH value 8.8) of 1/3 solution A volume, the 5mol/L KAc solution of 1/3 solution A volume, fully mix, room temperature is placed 15-30min, 4 ℃, the centrifugal 15min of 12000g, and water intaking phase transition, to new centrifuge tube, obtains solution B;
3) in solution B, add and the isopyknic chloroform of solution B, turn upside down two-phase is mixed, place on ice after 10min in 4 ℃, the centrifugal 10min of 12000g, get supernatant and transfer in new centrifuge tube;
Repeating step 3) chloroform extraction steps 1 time, obtains solution C;
4) in solution C, add and the isopyknic Virahol of solution C, mix to be placed in-20 ℃ and place 30min, 4 ℃, the centrifugal 15min of 12000g obtain RNA throw out;
5) use 75%(v/v) ethanol cleans RNA throw out 2 times, dries up the precipitation after washing, uses DEPC-ddH
2o dissolves the precipitation after drying up, and obtains the total RNA of Rhizoma Alpiniae Officinarum, in-80 ℃, saves backup.
The present invention is directed to the particularly feature of poly-hydroxy flavonol rich content of Rhizoma Alpiniae Officinarum secondary metabolites, on conventional guanidinium isothiocyanate-phenol-chloroform extraction process basis, carried out many-sided improvement:
Acetone, soluble poly V-Pyrol RC (PVP, K40) and beta-mercaptoethanol in RNA extract, have been added.Acetone can dissolve the coloring matters such as flavonols, water-soluble PVP can and form inner complex with the abundant combination of aldehydes matter, strong reductant beta-mercaptoethanol, reductive condition can be provided, polyphenols is difficult for oxidized, 3 kinds of materials act synergistically, and have effectively suppressed interference and the obstruction of polyphenols;
The present invention processes with the saturated phenol of Tris and high density acetic acid potassium after acquisition RNA slightly puies forward precipitation, can remove Partial Protein and polysaccharide simultaneously.
The present invention has following advantage and effect with respect to prior art:
The present invention utilizes the cracking extracting solution of improvement directly to grind with tissue, both can save the extracting time, again can extract early stage effectively stop tissue in phenolic compound and other secondary metabolites disturb and hinder; The guanidinium isothiocyanate of high density can deactivation RNA enzyme, grinds together with organizing, and in RNA discharges, can prevent the degraded of RNA enzyme to it; After obtaining RNA crude extract, by the saturated phenol of Tris and Potassium ethanoate combination treatment, can remove albumen and polysaccharide simultaneously, and cause the K of RNA liquid phase middle and high concentration
+, be conducive to chloroform extracting subsequently.Finally by obtaining high yield, high-quality RNA after washing with alcohol; The present invention is simple to operate, and extraction cost is low, reproducible.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis detected result that the total RNA of the fresh young leaflet tablet of Rhizoma Alpiniae Officinarum extracts.
Fig. 2 is the result figure that agarose gel electrophoresis detects the 18S rRNA reference gene that increases from Rhizoma Alpiniae Officinarum cDNA; Swimming lane 1 is DL-2000DNAmarker, and swimming lane 2 is the fresh young leaflet tablet cDNA of Rhizoma Alpiniae Officinarum amplification, and swimming lane 3 is Rhizoma Alpiniae Officinarum liquid nitrogen flash freezer leaf cDNA amplification.
Fig. 3 is the agarose gel electrophoresis detected result figure that the total RNA of the fresh young leaflet tablet of Rhizoma Alpiniae Officinarum extracts; Be from left to right: polysaccharide polyphenol plant RNA extraction test kit method, Trizol extracting solution extraction method, DL-2000DNAmarker.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
The preparation of embodiment medicine used and experimental article are processed:
1, the preparation of experimental drug:
Cracking extract preparation (10mL): add 5.85g guanidinium isothiocyanate, 0.8g soluble poly V-Pyrol RC (PVP, K40) in the CSB Extraction buffer of 6.6mL improvement, mix rear 65 ℃ of heating hydrotropies until dissolve completely, use DEPC-dd H
2o is settled to 10mL.
The CSB Extraction buffer of improvement forms: 25mmol/L Trisodium Citrate (PH4.8), 0.83%(w/v) sarcosyl, 4%(v/v) beta-mercaptoethanol.
DEPC-H
2o:100mL ddH
2in O, add diethylpyrocarbonate (DEPC) 1mL, 37 ℃ of shaken overnight, 1.05kg/cm
2autoclaving, 4 ℃ save backup.
2, experimental article is processed: plastic centrifuge tube and rifle head are all used 0.1%DEPC-ddH
2o soaks, then 1.05kg/cm
2sterilizing 20mim; Electrophoresis chamber and comb hydrogen peroxide dipping 30min, then use aseptic water washing, finally use 0.1%DEPC-ddH
2o rinses; 250 ℃ of continuous baking 8h of mortar and other glasswares and metalware.
The total RNA of the fresh young leaflet tablet of Rhizoma Alpiniae Officinarum extracts, and comprises the following steps:
(1) get 300mg Rhizoma Alpiniae Officinarum young leaflet tablet, put into the mortar of prior precooling, in mortar, add 1.5mL precooling cracking extract, the pre-cold acetone of 500 μ L, repeatedly add liquid nitrogen grinding to become powder, room temperature is placed 40min, the homogenate after grinding is transferred in 2mL centrifuge tube to 4 ℃, the centrifugal 15min of 12000g, shift colourless supernatant layer to another new centrifuge tube, obtain solution A; Described cracking extract is comprised of following component: 5mol/L guanidinium isothiocyanate, 25mmol/L Trisodium Citrate, 0.5%(w/v) sarcosyl, 4%(w/v) soluble poly V-Pyrol RC (PVP, K40), 4%(v/v) beta-mercaptoethanol;
(2) in the solution A obtaining in step (1), add the saturated phenol of Tris (pH value 8.8) of 1/3 volume, the 5mol/L KAc solution of 1/3 volume, fully mix, room temperature is placed 15～30min, 4 ℃, the centrifugal 15min of 12000g, water intaking phase transition, to new centrifuge tube, obtains solution B;
(3) in the solution B obtaining in step (2), add isopyknic chloroform, turn upside down two-phase is mixed, place on ice after 10min in 4 ℃, the centrifugal 10min of 12000g, get supernatant and transfer in new centrifuge tube; Chloroform extraction steps in repetition (3) 1 time, obtains solution C;
(4) in step (3) obtains solution C, add equal-volume Virahol, mix to be placed on and in-20 ℃, place 30min, 4 ℃, the centrifugal 15min of 12000g obtain RNA throw out;
(5) use 75%(v/v) ethanol cleaning step (4) RNA throw out is 2 times, dries up the precipitation after washing, uses DEPC-ddH
2o dissolves the precipitation after drying up, and obtains the total RNA of Rhizoma Alpiniae Officinarum, in-80 ℃, saves backup.
RNA integrity detection:
Get RNA2 μ L prepared by aforesaid method, 1% agarose gel electrophoresis (damping fluid is 0.5 * TBE) detects its integrity.By gel imaging, can observe well normal, 28S RNA, 18S RNA band are neat, bright, without assorted band, without hangover, show that the RNA extracting is complete, without DNA pollute, without degraded (see figure 1).
RNA quality examination:
Get RNA1 μ L prepared by aforesaid method, (Nanadrop Thermo Scientific on ultraviolet spectrometer, Wilmington, USA) in sample table, measure the ratio of A260 value and A260/A280, analyze RNA concentration and RNA quality, its A260/A280=1.99, output is 178 μ g/g fresh weights, shows to have high Quality and yield with the RNA that present method is extracted at the tender Rhizoma Alpiniae Officinarum blade of children.
The total RNA of liquid nitrogen flash freezer Rhizoma Alpiniae Officinarum blade extracts, and comprises the following steps:
(1) 200mg Rhizoma Alpiniae Officinarum young leaflet tablet is after liquid nitrogen flash freezer, put into the mortar of prior precooling, in mortar, add 1.0mL precooling cracking extract, the pre-cold acetone of 500 μ L, repeatedly add liquid nitrogen grinding to become powder, room temperature is placed 30min, homogenate is transferred in 2mL centrifuge tube to 4 ℃, the centrifugal 15min of 12000g, shift colourless supernatant layer to another new centrifuge tube, obtain solution A;
(2) in the solution A obtaining in step (1), add the saturated phenol of Tris (pH value 8.8) of 1/3 volume, the 5mol/L KAc solution of 1/3 volume, fully mix, room temperature is placed 15～30min, 4 ℃, the centrifugal 15min of 12000g, water intaking phase transition, to new centrifuge tube, obtains solution B;
(3) in the solution B obtaining in step (2), add isopyknic chloroform, turn upside down two-phase is mixed, place on ice after 10min in 4 ℃, the centrifugal 10min of 12000g, get supernatant and transfer in new centrifuge tube; Chloroform extraction steps in repetition (3) 1 time, obtains solution C;
(4) in step (3) obtains solution C, add equal-volume Virahol, mix to be placed in-20 ℃ and place 30min, 4 ℃ of centrifugal 15min of 12000g obtain RNA throw out;
(5) use 75%(v/v) ethanol cleaning step (4) RNA throw out is 2 times, dries up the precipitation after washing, uses DEPC-ddH
2o dissolves the precipitation after drying up, and obtains the total RNA of Rhizoma Alpiniae Officinarum, in-80 ℃, saves backup.
Application the present embodiment, obtaining liquid nitrogen flash freezer Rhizoma Alpiniae Officinarum blade RNA productive rate is 124 μ g/g fresh weights (total RNA amount/sample sizes), through agarose gel electrophoresis result, show that RNA integrity is good, on uv-spectrophotometric instrument, measure 260nm and 280nm absorbance ratio A260/A280 is 2.03, can meet molecular biology experiment needs.
The amplification of the cDNA reverse transcription of the total RNA of Rhizoma Alpiniae Officinarum and reference gene 18S rRNA gene, comprises the following steps:
(1) in PCR pipe, prepare following template ribonucleic acid: in PCR pipe, add the total RNA(1.24 μ of 2 μ L Rhizoma Alpiniae Officinarum quick-frozen blade g) or the total RNA(1.78 μ of fresh blade g), 1 μ L Oligo(dT) 15primer(0.5 μ g), 7 μ L DEPC-dd H
2o, mixes rear 70 ℃ of constant temperature 10min, places 2min on ice, and the centrifugal several seconds makes solution aggregation in the pipe end;
(2) in step (1) PCR pipe, add following reaction solution: 2 μ L M-MLV5 * Reaction Buffer, 0.5 μ L dNTP(10mM each), 0.25 μ L Ribolock RNase Inhibitor, 0.5 μ LM-MLV RT, 1.75 μ L DEPC-ddH
2p, after mixing, in 42 ℃ of reaction 1.5h, taking-up is placed in 70 ℃ of heating 5min and makes reversed transcriptive enzyme inactivation, obtains cDNA after cooled on ice;
(3) getting the fresh or liquid nitrogen flash freezer Rhizoma Alpiniae Officinarum young leaflet tablet reverse transcription cDNA of 1 μ L is pcr template, amplification plant reference gene 18S rRNA Gene Partial sequence;
(4) get 5 μ L products and carry out electrophoresis in 1.2%TBE sepharose (damping fluid is 0.5 * TBE), the results are shown in Figure 2, in fresh or liquid nitrogen flash freezer Rhizoma Alpiniae Officinarum young leaflet tablet reverse transcription cDNA all amplification to bright, with expect the big or small reference gene 18S rRNA Gene Partial sequence (～300bp) conforming to, and the target stripe increasing from the high fresh leaf cDNA of RNA concentration is the target stripe of bright amplification in the liquid nitrogen flash freezer leaf cDNA low from RNA concentration obviously, shows to adopt total RNA that the present invention extracts to be suitable for carrying out RT-PCR equimolecular biological experiment.
In this embodiment, the synthetic agents useful for same of cDNA is and adopts Takala company product;
Reference gene 18S rRNA amplimer is:
18s-F：5′-ATTCCTAGTAAGCGCGAGTCATCAG；
18s-R：5′-CAATGATCCTTCCGCAGGTTCAC；
PCR reaction system is (25 μ L): ddH
2o12.5 μ L, Taq PCR MasterMix(ZOMANBIO) 9.5 μ L, each 1 μ L of positive and negative primer, cDNA1 μ L;
PCR reaction conditions is: 94 ℃ of denaturation 3min, and 94 ℃ of sex change 30s, 55 ℃ of annealing 40s, 72 ℃ are extended 40s, 25 circulations, 72 ℃ are extended 10min eventually.
Comparative example 1
CTAB(cetyl trimethylammonium bromide) method is extracted the total RNA of Rhizoma Alpiniae Officinarum blade
Reference literature (Yang Hong etc., are designated as basic RT-PCR technology for detection Strawberry mottle virus in utilization, Plant Pathology, and 2005,35(2): method 116-122) is extracted, and concrete operations are as follows:
(1) first at 3mL, contain (2%CTAB in the Extraction buffer of CTAB; 100mmol/L TrisHCl, pH value 8.0; 1.4mol/L NaCl; 20mmol/L EDTA, pH value 8.0) add 60 μ L mercaptoethanols, put 65 ℃ of preheatings, then 600mg is put into wherein through the Rhizoma Alpiniae Officinarum young leaflet tablet powder of liquid nitrogen grinding, after firmly mixing, be placed in 65 ℃ and be incubated 1h; Then take out sample, naturally cool to room temperature; With isopyknic chloroform/primary isoamyl alcohol mixed solution (wherein the volume ratio of chloroform and primary isoamyl alcohol is 24:1) extracting to without interface without white protein layer, the centrifugal 20min of 10000g under room temperature;
(2) get supernatant, add 10mol/L LiCl to final concentration be 3mol/L; In-20 ℃ of placements, spend the night, through 16000g, 4 ℃ of centrifugal 20min;
(3) remove supernatant liquor, 1mL DEPC water dissolution for precipitation, then use isopyknic phenol/chloroform/primary isoamyl alcohol mixed solution (volume ratio of phenol, chloroform, primary isoamyl alcohol is 25:24:1) extracting; Get supernatant liquor, then through chloroform/primary isoamyl alcohol mixed solution (24:1) extracting; Get supernatant, by 1/30 volume, add 3mol/L NaAc(pH value 5.2) and the dehydrated alcohol of 1/10 volume, mix rear ice bath 30min, 16000g, 4 ℃ of centrifugal 20min; Get supernatant liquor, by 1/10 volume, add NaAc(3mol/L, pH value 5.2) and the dehydrated alcohol of 3 times of volumes, place 4h at-70 ℃; Through 16000g4 ℃ of centrifugal 20min, 75% ethanol drip washing 2 times for throw out, is dissolved in the DEPC water of appropriate volume after being dried;
(4) get step (3) DEPC water dissolution thing 2 μ L and in 1% agarose gel electrophoresis, detect the RNA of its extracting.
Adopt present method, agarose gel electrophoresis detects does not have band to occur, shows that the method fails to extract RNA from Rhizoma Alpiniae Officinarum young leaflet tablet.
Comparative example 2
N – sarcosyl (LSS) method is extracted the total RNA of Rhizoma Alpiniae Officinarum blade
Reference literature (Wu Jianyang etc., rapidly and efficiently extract the novel method that RNA is organized in lichee young fruit and abscission zone, gardening journal, and 2011,38(6): method 1191) is extracted, and its operating process is as follows:
(1) fresh Rhizoma Alpiniae Officinarum young leaflet tablet 300mg is placed in to liquid nitrogen grind into powder, after becoming dry powder by liquid nitrogen grinding, pack 2mL centrifuge tube into, add again 1mL to be preheating to the extracting solution of 70 ℃, acutely rocking centrifuge tube makes sample mix even, room temperature is jiggled 5min subsequently, 25 ℃, after the centrifugal 15min of 12000r/min, supernatant liquor is proceeded to new centrifuge tube; Extracting solution used is (100mol/L Tris, 200mol KCl/L, 15mol/L EDTA, 4%(w/v) N – sarcosyl (LSS), with KOH adjust pH to 9.0; Before use, add 2%(w/v) PVP), in 1mL extracting solution, add the DTT(dithiothreitol (DTT) of 15 μ L) and ratio (10mol/L) adds DTT in extracting solution;
(2) to being equipped with in the centrifuge tube of supernatant liquor, add 200 μ L3mol/L NaAc(pH values 5.2), acutely rock centrifuge tube, mix latter 25 ℃ completely, the centrifugal 5min of 12000r/min.Supernatant liquor is proceeded in new centrifuge tube;
(3) to being equipped with in the centrifuge tube of supernatant liquor, add 30 ℃ of 1mL(Cun Fang Yu –) Virahol, shake up latter 25 ℃, the centrifugal 10min of 12000r/min, abandons supernatant liquor;
(4) by 400 μ L Tris-HCl(pH values 7.5) dissolution precipitation, then add 100 μ L3mol/L NaAc(pH values 5.2), 25 ℃, the centrifugal 5min of 12000r/min, proceeds to supernatant liquor in new centrifuge tube;
(5) in new centrifuge tube, add 1.2mL precooling dehydrated alcohol, the centrifuge tube that turns upside down gently mixes 30 ℃ of Hou ， – and puts 30min, and 25 ℃, the centrifugal 10min of 12000r/min, collecting precipitation;
(6) by 75% washing with alcohol precipitation of 30 ℃ of precoolings of 1.5mL –, 25 ℃, the centrifugal 10min of 10000r/min, abandons supernatant liquor, repeats 1 time, vacuumizes rear use 30 μ L sterilized waters and dissolves;
(7) get step (6) water dissolution thing 2 μ L and in 1% agarose gel electrophoresis, detect the RNA of its extracting.
Adopt present method, agarose gel electrophoresis detects does not have band to occur, shows that the method fails to extract RNA from Rhizoma Alpiniae Officinarum young leaflet tablet.
Comparative example 3
The extracting of guanidinium isothiocyanate-chloroform, the saturated phenol method of purification of Tris are extracted the total RNA of Rhizoma Alpiniae Officinarum blade
With reference to patent < <, from be rich in the plant tissue of polysaccharide polyphenol and secondary metabolites, extract total RNA method > > (publication number: method CN101638651B), concrete operations are as follows:
(1) take fresh Rhizoma Alpiniae Officinarum young leaflet tablet 300mg and be placed in liquid nitrogen grind into powder, proceed in 2mL centrifuge tube, add rapidly the RNA extraction buffer of 1mL precooling, mix and be placed on 5min on ice, the 3mol/L sodium-acetate 1mL that adds again pH value 4.5, turns upside down and mixes; RNA extraction buffer moiety is 4mol/L guanidinium isothiocyanate, 25mmol/L Trisodium Citrate, and 0.5%w/v sarcosyl, 2%w/v soluble poly V-Pyrol RC, before using, every 100mL extraction buffer adds 0.5mL beta-mercaptoethanol;
(2) add the chloroform of 1mL, concuss mixes two-phase, places on ice after 10min in 4 ℃, the centrifugal 10min of 12000g, gets supernatant liquor and is placed in new centrifuge tube;
(3) the chloroform extraction steps in repetition (2) is 1 time;
(4) in supernatant liquor, add approximately 1/3 Virahol, mix and be placed on-20 ℃ of 30min, then 4 ℃ of centrifugal 15min of 12000g obtain RNA precipitation;
(5) by 75% ethanolic soln washing and precipitating 2 times, be placed in drying at room temperature 20min, the ddH processing with DEPC
2o thoroughly dissolves RNA precipitation;
(6) in RNA solution, add the saturated phenol of Tris of approximately 1/3 volume, concuss mixes two-phase, and the centrifugal 10min of normal temperature 12000g, gets supernatant liquor;
(7) in supernatant liquor, add approximately 1/3 volume chloroform, after concuss mixes, the centrifugal 10min of normal temperature 12000g, gets supernatant liquor;
(8) in supernatant liquor, add 3mol/L sodium-acetate 0.1-0.2mL and the 1mL Virahol of pH value 4.5-5.5, after mixing, in-20 ℃, place 30min, 4 ℃, the centrifugal 15min of 12000g, wash throw out 2 times with 75% ethanolic soln, after drying at room temperature 20min, the ddH processing with 20 μ L DEPC
2o dissolution precipitation;
(9) get step (8) DEPC water dissolution thing 2 μ L and in 1% agarose gel electrophoresis, detect the RNA of its extracting.
Adopt present method, agarose gel electrophoresis detects does not have band to occur, shows that the method fails to extract RNA from Rhizoma Alpiniae Officinarum young leaflet tablet.
Comparative example 4
Trizol reagent solution method is extracted the total RNA of Rhizoma Alpiniae Officinarum blade, comprises the following steps:
Fresh Rhizoma Alpiniae Officinarum young leaflet tablet 300mg is placed in to liquid nitrogen grind into powder, proceed in the centrifuge tube of processing through diethylpyrocarbonate (DEPC), add 1.0mL Trizol RNA extracting test solution (Shanghai Sheng Gong bio-engineering corporation), mix the centrifugal 15min of 5000rpm under rear thermal agitation 5min room temperature; Subsequent step is with embodiment 2.
Utilize present method, the RNA extracting all degrade (the middle swimming lane of Fig. 3).
Comparative example 5
Invitrogen test kit extracts the total RNA of Rhizoma Alpiniae Officinarum blade
Use the plant total RNA extraction reagent box of Invitrogen company, concrete operations by specification.
Adopt present method, fail to extract RNA from Rhizoma Alpiniae Officinarum young leaflet tablet.
Comparative example 6
The raw work test kit in Shanghai extracts the total RNA of Rhizoma Alpiniae Officinarum blade
Use the polysaccharide polyphenol plant tissue RNA that Shanghai Sheng Gong bio-engineering corporation produces to extract the test kit of development, concrete operations by specification.
Utilize present method, the RNA severely degrade extracting (left side swimming lane of Fig. 3).
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (2)
1. an extracting method of the total RNA of Rhizoma Alpiniae Officinarum, is characterized in that comprising the following steps:
1) get Rhizoma Alpiniae Officinarum young leaflet tablet and put into precooling mortar, in mortar, add precooling cracking extract and pre-cold acetone, repeatedly add liquid nitrogen grinding to become powder, room temperature is placed 30-40min, gained homogenate is transferred in centrifuge tube, 4 ℃, the centrifugal 15min of 12000g, shift colourless supernatant layer to another centrifuge tube, obtains solution A;
Described cracking extract is comprised of following component: 5mol/L guanidinium isothiocyanate, 25mmol/L Trisodium Citrate, 0.5%(w/v) sarcosyl, 4%(w/v) soluble poly V-Pyrol RC, 4%(v/v) beta-mercaptoethanol;
2) in solution A, add the saturated phenol of Tris of 1/3 solution A volume, the 5mol/LKAc solution of 1/3 solution A volume, fully mix, room temperature is placed 15-30min, 4 ℃, the centrifugal 15min of 12000g, and water intaking phase transition, to new centrifuge tube, obtains solution B;
3) in solution B, add and the isopyknic chloroform of solution B, turn upside down two-phase is mixed, place on ice after 10min in 4 ℃, the centrifugal 10min of 12000g, get supernatant and transfer in new centrifuge tube;
Repeating step 3) chloroform extraction steps 1 time, obtains solution C;
4) in solution C, add and the isopyknic Virahol of solution C, mix to be placed in-20 ℃ and place 30min, 4 ℃, the centrifugal 15min of 12000g obtain RNA throw out;
5) use 75%(v/v) ethanol cleans RNA throw out 2 times, dries up the precipitation after washing, uses DEPC-ddH
2o dissolves the precipitation after drying up, and obtains the total RNA of Rhizoma Alpiniae Officinarum;
Step 2) the described saturated phenol of Tris, its pH value is 8.8.
2. the extracting method of the total RNA of Rhizoma Alpiniae Officinarum according to claim 1, is characterized in that: described Rhizoma Alpiniae Officinarum blade is liquid nitrogen flash freezer young leaflet tablet or fresh young leaflet tablet.
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| CN106801051A (en) * | 2017-03-09 | 2017-06-06 | 中国科学院华南植物园 | A kind of kit and extracting method for extracting plant RNA |
| CN108997510A (en) * | 2018-08-09 | 2018-12-14 | 海南医学院 | A kind of anti-oxidant application of galangal polysaccharide and its isolation and purification method |
| CN110760508A (en) * | 2019-05-08 | 2020-02-07 | 漯河市农业科学院 | Method for extracting total RNA of wheat |
| EP4163369A1 (en) | 2021-10-08 | 2023-04-12 | AXAGARIUS GmbH & Co. KG | Use of mixtures of polyvinylpyrrolidone and ammonium sulfate in the isolation of nucleic acids |
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| CN106801051A (en) * | 2017-03-09 | 2017-06-06 | 中国科学院华南植物园 | A kind of kit and extracting method for extracting plant RNA |
| CN108997510A (en) * | 2018-08-09 | 2018-12-14 | 海南医学院 | A kind of anti-oxidant application of galangal polysaccharide and its isolation and purification method |
| CN110760508A (en) * | 2019-05-08 | 2020-02-07 | 漯河市农业科学院 | Method for extracting total RNA of wheat |
| EP4163369A1 (en) | 2021-10-08 | 2023-04-12 | AXAGARIUS GmbH & Co. KG | Use of mixtures of polyvinylpyrrolidone and ammonium sulfate in the isolation of nucleic acids |
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