CN102805078B - Short-term preservation method of rubber tree leaves for RNA (ribonucleic acid) extraction - Google Patents
Short-term preservation method of rubber tree leaves for RNA (ribonucleic acid) extraction Download PDFInfo
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- CN102805078B CN102805078B CN 201210297803 CN201210297803A CN102805078B CN 102805078 B CN102805078 B CN 102805078B CN 201210297803 CN201210297803 CN 201210297803 CN 201210297803 A CN201210297803 A CN 201210297803A CN 102805078 B CN102805078 B CN 102805078B
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Abstract
The invention relates to a short-term preservation method of rubber tree leaves for RNA (ribonucleic acid) extraction. The short-term preservation method of rubber tree leaves for RNA extraction includes: collecting tender leaves of rubber tree in light bronze-colored stage, deep bronze-colored stage, discoloring stage or light green stage, loading the leaves in a centrifugal tube, adding storage buffer, placing the centrifugal tube in an ice box or ice barrel containing ice cubes at 0-4 DEG C, bringing the box or barrel to a lab, and placing the box or barrel on an ice rock at 0-4 DEG C for standby. Tissues of rubber tree leaves are stored by the storage buffer and low-temperature storage, so that freshness of the rubber tree leaves can be maintained in a short time effectively and remote sampling and material transport and storage are facilitated. The short-term preservation method has the advantages of simple process, low cost, effectiveness and the like, and quality security in sample RNA extraction is improved.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of leaf tissue store method, specifically is the bamboo grows blade short-term store method that a kind of RNA of being used for extracts.
Background technology
Bamboo grows (Hevea spp.) is the perennial cross pollination arbor of Euphorbiaceae (Euphorbiaceae) Hevea (Hevea), for the extraordinary forest that glue is produced in the torrid areas, is the important economic crops of a class.China has had the natural rubber based ground of scale and mainly has been distributed in Hainan, Yunnan and Guangdong 3 provinces.Since the nineties in last century, develop rapidly along with molecular biological, Studies on Hevea Brasiliensis also progresses into molecular level.From the transcriptional level to bamboo grows output, degeneration-resistant, grow and related gene expression such as metabolism and regulation and control are studied, be that bamboo grows is expressed quantitative trait locus (expression quantitative traitlocus, eQTL) a kind of important means of work such as location, transgenic breeding, transformed variety evaluation, assistant breeding is the important foundation of carrying out isolated genes and the biological study of analyzing gene expression characterization equimolecular and obtain high-quality total RNA.
At present, the conventional method of extracting bamboo grows blade geneome RNA mainly contains hot boric acid/Proteinase K method and CTAB/ acid phenol method, and two kinds of methods are proposed the requirement that RNA concentration and purity all can reach follow-up cDNA library construction or pcr amplification equimolecular biological experiment.Ionic detergent SDS, DOC and CTAB are by making protein denaturation, destroy membrane structure and untie the protein that is connected with nucleic acid, thereby realize that nucleic acid is free in the cracking system.But higher, the better quality of purity of the RNA that extracts with hot boric acid/Proteinase K method, reason is that the bamboo grows tissue is rich in aldehydes matter, and the RNA extract of selecting the borate buffer liquid system for use helps removing the aldehydes matter in the tissue, boric acid wherein can rely on hydrogen bond to form compound with phenolic compound, suppressed aldehydes matter oxidation and with the combining of RNA.
In the research of extracting plant leaf blade RNA, conventional method adopts fresh or freezing material more.The material that extracts RNA should keep fresh as far as possible, and the quality that sample is preserved will directly influence the extraction effect of RNA.Preferably carry liquid nitrogen container, dry ice chest, ice chest or ice bucket when therefore plantation is gathered fresh material, but in real work, sometimes because remote sampling can't be carried liquid nitrogen container, and the shortcoming of portable dry ice chest, ice chest or ice bucket is the weak point of holding time, can not satisfy the needs of sampling, this moment, the preservation of plant sample became the committed step whether being kept perfectly of structure of its RNA of decision.
After transporting the laboratory back, preferably in time extracts by the sample that satisfies test requirements document RNA, sometimes the arrangement in the test can't in time be extracted RNA to fresh material, freezing is to keep one of effective and efficient manner of plant sample structural constituent, the at present freezing ultralow temperature that mainly concentrates on is preserved, and quality and the quantity of total RNA that the sample of-80 ℃ of refrigerator preservations is carried are all suitable with fresh blade.But the field carry the sampling of liquid nitrogen or dry ice exist cost an arm and a leg, tank body heaviness, complex operation, volatile, dangerous high shortcoming, fail to reach effect such as simple, economic, safe, efficient.
Summary of the invention
The objective of the invention is to provide a kind of bamboo grows blade short-term store method of the RNA of being used for extraction at the deficiencies in the prior art, its method of utilizing the storage buffer solution to combine with deepfreeze is preserved the bamboo grows leaf tissue, can realize effectively that the short time keeps the freshness of bamboo grows blade, be convenient to remote sampling, transport of materials and storage, improved the safety of sample RNA extraction quality effectively.
The technology used in the present invention principle: borate buffer solution (sodium tetraborate, Sodium Borate, SBX, PH9.0) provide a buffer environment, prevent that nucleic acid is destroyed, studies show that, extract the boric acid that contains in the buffer solution and under weak basic condition, can form compound with hydrogen bond with phenols, suppress aldehydes matter oxidation and with the combining of RNA, have anti-rot fungi simultaneously, moth acts on, the concentration that is used for rubber wood timber is 30～50mM; EGTA chelating Mg
2+Or Mn
2+Ion suppresses the RNase activity; Polyvinylpyrrolidone (PVP) is a kind of high molecular synthetic resin, water soluble, ethanol and chloroform, industrial fining agent, the stabilizing agent of being commonly used for, energy complexing polyphenols, prevent that polyphenol substance is oxidized to quinones substance, avoid the solution browning and have antioxidation; Dithiothreitol (DTT) (DTT) is a reductant commonly used, have the antioxidation same with beta-mercaptoethanol, can prevent the oxidation of aldehydes matter in the bamboo grows blade effectively, avoid brown stain, help the removal of phenol, but DTT compares with beta-mercaptoethanol, the penetrating odor of DTT is much smaller, and toxicity is also much lower than beta-mercaptoethanol, and DTT is during than low 7 times of the concentration of beta-mercaptoethanol, both effects are close, and only the DTT price is slightly higher.Sucrose (Sucrose) is for blade provides carbon source, and the excised leaf autophyting ability is relatively poor, just can influence its physiological metabolism unavoidably, and sucrose will resolve into glucose and the fructose form that blade cell can directly absorb behind infusion and autoclaving.
The technical solution adopted in the present invention:
A kind of bamboo grows blade short-term store method that is used for the RNA extraction, be to gather the bamboo grows tender leaf that is in little bronze phase, big bronze phase, variable color phase or pale green phase, put into 10～50ml centrifuge tube, divide and add 5～25ml storage buffer solution for 2 times, then centrifuge tube is put into that ice cube, temperature are housed is 0～4 ℃ ice chest or ice bucket, after taking back the laboratory, be shelved on temperature and be on 0～4 ℃ the ice cube, stand-by.Described storage buffer solution is to join in the sterile water Sodium Borate, Na-EGTA, PVP-40, DTT and sucrose formulated, wherein each components contents is: Sodium Borate 150～250mM, Na EGTA 7.5～12.5mM, PVP 2～4%(W ∕ V), DTT 10～20mM, sucrose 3～5%(W ∕ V), wherein PVP, cane sugar content are mass volume ratio content.
Determine the sample treatment mode according to retention cycle length:
1, short-term is preserved: need sample cell is packed in the sealed bag, write label, treat all samples unified placement that dispose.The research of carrying out in the strange land for test arrangement, sending of material is an indispensable link, at the experiment of on rna level, carrying out genomics research,, cover chest completely as long as above-mentioned ready sample is placed in the bubble chamber that has been equipped with ice bag (0～4 ℃), use rubber belt sealing, even delay on the road, hot weather, ice bag also dissolves, after one week, it is green that the sample of receiving still keeps.
2, long preservation: remove to store buffer solution, screw and manage lid, put into the container that fills liquid nitrogen, this processing mainly is that protection RNA does not degrade, and treats-70～-80 ℃ of refrigerators of all samples unified placement in abundant freezing back; Sample can be taken at any time, possesses to carry the same effect of fresh blade that-80 ℃ of liquid nitrogen samplings are preserved or liquid nitrogen frozen is crossed.
The method that the present invention utilizes the storage buffer solution to combine with deepfreeze is preserved the bamboo grows leaf tissue, can realize effectively that the short time keeps the freshness of bamboo grows blade, be convenient to remote sampling, transport of materials and storage, have characteristics such as technology is simple, cost is low, effective, improved the safety of sample RNA extraction quality.
Description of drawings
Fig. 1 is the total RNA electrophoresis pattern of bamboo grows leaf tissue that utilizes hot boric acid/Proteinase K method to extract.
Among the figure: 1 is fresh blade, and 2 for adopting the inventive method short-term to preserve blade.
Fig. 2 is the total RNA effect detection of the bamboo grows blade figure that utilizes CTAB/ acid phenol method to extract.
Among the figure: 1 carries the liquid nitrogen sampling after the blade of ultralow temperature long preservation for adopting, and 2 for adopting the present invention to store the preservation of buffer solution short-term and liquid nitrogen flash freezer after the blade of ultralow temperature long preservation.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
One, buffer solution allotment
For improving the quality of storage buffer solution, buffer solution needs allotment, and five principal elements in the prescription are respectively tested (seeing Table 1) by three levels.Seek the horizontal combination of the best, guarantee that buffer solution is effective, economical.Adopt this assembly side's preparation leaf tissue to preserve the storage buffer solution.After the storage buffer solution preparation, steam sterilization 15min under 121 ℃ high pressure, but because of DTT or the solution that contains DTT can not carry out HIGH PRESSURE TREATMENT, DTT preparation back filtration sterilization, PVP-40 and DTT face with preceding adding and store buffer solution.
Table 1 storage buffer solution five factor Orthogonal Experiment and Design tables
Two, blade is preserved
The tender leaf that collects is put into 10～50ml centrifuge tube, add 5～25ml storage buffer solution, put into ice chest or the ice bucket (0～4 ℃) carried, after taking back the laboratory, remove to store buffer solution, screw the pipe lid, put into the container quick-frozen 3～5min that fills liquid nitrogen, place-70～-80 ℃ of refrigerator long preservation at last.The sample that need send replenishes earlier a certain amount of storage buffer solution, screws the pipe lid, and sample cell is packed in the sealed bag, write label, treat that all samples unified placement that dispose has been equipped with in the bubble chamber of ice bag, covers chest completely, use rubber belt sealing, external application carton bag is real, and strap binds.
Three, RNA extracts
Embodiment one
1. will from storage buffer solution and refrigerator (70 ℃～-80 ℃), take out the fresh blade of 1g respectively through the sample of short-term or long preservation, blade will be put into the precooling mortar, grind to form fine powder in the liquid nitrogen, change in two 5ml centrifuge tubes.(just needing 1 5ml centrifuge tube about 0.5g)
2. add 2.5ml in each 5ml pipe and be preheating to 80 ℃ extraction buffer solution (200mM Sodium Borate PH9.0,30mM EGTA, 1% SDS, 10mM DTT, 1% DOC, 2% PVP-40), even oar 2 min.(DEPC-H is used in even oar rotary head earlier
2O washes several times more, at least 3 times)
3. will spare oar liquid and change in the new centrifuge tube that is added with 200 μ l 20mg/ml Proteinase Ks, in 42 ℃ of shaking tables, bathe 1.5h with 100rpm rotating speed temperature.
4. add 500 μ l 1.6mol/L KCl, to final concentration 160mmol/L, ice bath 1h.
5. 4 ℃, the centrifugal 20min of 12000rpm, (or 4 ℃, the centrifugal 30min of 10000rpm) shift supernatant to another new pipe, abandon precipitation.
6. add 1/2 volume 6mol/L LiCl in the supernatant, to final concentration 2mol/L ,-20 ℃ of precipitation 2h.
7. 4 ℃, the centrifugal 20min of 12000rpm keep precipitation, abandon supernatant.
8. the RNA precipitation is washed twice, 4 ℃, the centrifugal 15min of 10000rpm with 70% ethanol is heavy, abandons supernatant.
9. 4 ℃, the centrifugal 5min of 12000rpm abandon supernatant.
10. the DEPC-H that adds 100 μ l after the vacuum drying again
2O makes it abundant dissolving.
11. 4 ℃, the centrifugal 10min of 12000rpm transfer to another new pipe with supernatant, abandon precipitation.
Detect the RNA quality 12. get 1 μ l with agarose electrophoresis, all the other RNA preserve down in-70 ℃.
Annotate: PVP-40 and DTT reagent are used preceding adding facing.
Embodiment two
1. field acquisition or the nonlocal sample of sending are taken out from foam box, or the sample that from-70～-80 ℃ of refrigerators, takes out, choose the fresh tender tissue of 800mg bamboo grows in mortar, add liquid nitrogen and fully be ground into powder, transfer in the 10ml centrifuge tube.
2. the RNA extract (1.4mol/L NaCl, 0.1mol/L TrisHCl (adding after the PH8.0 sterilization), 20mmol/L EDTA, 2% CTAB, 2% PVP, 1% (V/V) β-Me(time spent add) that adds 4ml in the centrifuge tube, above-mentioned except that TrisHCl first 0.1% DEPC-H that use of all the other medicines
212hs is rocked in the O preparation, and sterilization adds 0.1mol/L Tris then again, regulates pH value to 8.0 with HCl, 320 μ l β-Me, vibration mixing, 65 ℃ of about 30min of water-bath, mixed 2-3 time midway.
3. the chloroform that adds 0.6 times of volume again, abundant back and forth mixing, ice bath 10min then.
4. 4 ℃, the centrifugal 20min of 9000rpm transfer to supernatant in another new 10ml centrifuge tube.
5. use the extracting of equal-volume chloroform more once, 4 ℃, the centrifugal 20min of 9000rpm.
6. it is to sterilize after 0.1% DEPC rocks 12hs that the 8mol/L LiCl(that adds 1/3 times of volume (900 μ l) in the supernatant adds final concentration) solution, evenly mixed, then-20 ℃ more than the 2h.
7. 4 ℃, the centrifugal 20min of 10000rpm discard top solution, and precipitation (is used 0.1% DEPC-H after sterilizing with 70% ethanol
2The O preparation) heavy washing, and transfer in the 2ml centrifuge tube.
8. the centrifugal 5min of 4000rpm inhales and removes ethanolic solution, with vavuum pump precipitation is drained.
9. the H that adds 1200 μ l
2The O-DEPC dissolution precipitation adds the 8mol/L LiCl solution of 1/3 times of volume (400 μ l) again, and is evenly mixed, then-20 ℃ more than the 1h.
10. 4 ℃, the centrifugal 20min of 9000rpm collect the RNA precipitation.
11. the RNA precipitation washs twice with 70% ethanol is heavy, adds the DEPC-H of 80 μ l after the vacuum drying again
2O makes it fully dissolving and gets 1 μ l and detect the RNA quality with agarose electrophoresis, and all the other RNA preserve down in-70 ℃.
Annotate: 240 ℃ of bakings of vessel 4hs, plastics equipment 0.1% DEPC-H
2O handles 24hs, 0.1% DEPC-H for preparing
2O sterilizes after rocking 2hs under 37 ℃.
Four, the mensuration of RNA purity and concentration
1, UV Absorption detects: get 1ul RNA and be diluted to 200ul, measure the light absorption value of total RNA at 260nm and 280nm place on ultraviolet specrophotometer, determine RNA purity according to A260/A280.Estimate the concentration of sample RNA according to the absorbance value of 260nm place mensuration.The results are shown in Table 2.
Table 2 RNA sample detection information
2, agarose gel electrophoresis detects: get the 1ul sample and carry out electrophoresis in 1.2% Ago-Gel that contains ethidium bromide (EB), electrophoretic buffer is 1 * TAE, and swimming speed is 7V/cm, and uviol lamp is observed the quality of RNA and molecular weight roughly down.
Ultraviolet spectrometer the analysis showed that the RNA from fresh blade respectively detects index and is respectively 28S:18S=1.4, OD260/280=2.03, OD260/230=2.06, this method is preserved the 28S:18S=1.5 of blade gained RNA, OD260/280=2.05, OD260/230=2.24, quality does not have significant difference between the two, no matter quality or quantity, two kinds of separate sources blades have all obtained to satisfy fully the sample of follow-up test requirement.Geneome RNA agarose gel electrophoresis testing result is seen Fig. 1, Fig. 2, the bamboo grows geneome RNA electrophoresis pattern of Fig. 1 for adopting hot boric acid/Proteinase K method to extract, the RNA effect detection figure of Fig. 2 for adopting CTAB/ acid phenol method to extract.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (1)
1. one kind is used for the bamboo grows blade short-term store method that RNA extracts, it is characterized in that gathering the bamboo grows tender leaf that is in little bronze phase, big bronze phase, variable color phase or pale green phase, put into 10～50ml centrifuge tube, divide and add 5～25ml storage buffer solution for 2 times, then centrifuge tube is put into that ice cube, temperature are housed is 0～4 ℃ ice chest or ice bucket, after taking back the laboratory, be shelved on temperature and be on 0～4 ℃ the ice cube, stand-by; Described storage buffer solution is to join in the sterile water Sodium Borate, Na-EGTA, PVP-40, DTT and sucrose formulated, wherein each components contents is: Sodium Borate 150～250mM, Na EGTA 7.5～12.5mM, PVP-40 2～4%, DTT 10～20mM, sucrose 3～5%, wherein PVP-40, cane sugar content are mass volume ratio content.
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| CN107873699B (en) * | 2017-11-27 | 2021-02-12 | 云南省热带作物科学研究所 | Preservation method of rubber leaves |
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