CN103589714A - Method for quickly and efficiently extracting wheat puccinia striiformis f.sp.tritici RNA (Ribonucleic acid) - Google Patents
Method for quickly and efficiently extracting wheat puccinia striiformis f.sp.tritici RNA (Ribonucleic acid) Download PDFInfo
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- CN103589714A CN103589714A CN201310548719.4A CN201310548719A CN103589714A CN 103589714 A CN103589714 A CN 103589714A CN 201310548719 A CN201310548719 A CN 201310548719A CN 103589714 A CN103589714 A CN 103589714A
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Abstract
RNA (Ribonucleic acid) is ubiquitous in living biological cells, is an intermediate vector of genetic information of organisms and takes part in DNA (deoxyribonucleic acid) transcription and protein synthesis. With the change of environmental factors, RNA can make a response at the first time, and isolated RNA or dead cell RNA is very easily degraded, so that RNA is taken as a first index for detecting if viable microorganisms are currently existent. Extracting complete and high-quality wheat puccinia striiformis f.sp.tritici urediospore RNA is a basis for further conducting a research of transcriptomics, puccinia striiformis f.sp.tritici survival amount monitoring and the like. Key technologies involved in a created method for extracting the wheat puccinia striiformis f.sp.tritici RNA comprise complete transfer of test materials and oxidation prevention. A key preprocessing solution for guaranteeing complete transfer of wheat puccinia striiformis f.sp.tritici and improving the grinding effect is 0.01 M Tris-HCl with the pH value of 8.0; involved main ingredients for preventing oxidation comprise 1% of SDS (W to V), 1% of beta-mercaptoethanol (V to V) and 16% of PVP (polyvinyl pyrrolidone) (W to V).
Description
Technical field: the present invention relates to a kind of wheat stripe rust RNA extracting method rapidly and efficiently.
Technical background: wheat stripe rust is obligate parasite, can not utilize substratum to carry out artificial culture, its propagulum uredospore, winter spore, haustorium, mycelium etc. are difficult to obtain, and the extraction of its wheat stripe rust uredospore RNA is subject to many conditionalities to seem more difficult.Traditional uredospore extracting method is to take the uredospore of tens grams to transfer to liquid nitrogen grinding in mortar.But find in leaching process, when the uredospore taking is poured in liquid nitrogen, uredospore is because very light weight is easily splashed, and a small part uredospore can be stained with on tube wall, can not transfer in mortar completely., after liquid nitrogen grinding, find during transferred material, the uredospore after grinding is easily stained with mortar bottom meanwhile, is difficult to scrape down completely.Several reasons usually cause a large amount of uredospores to be lost in leaching process above.At present, wheat stripe rust RNA extracts the methods that adopt test kit more, but cost is very high.
We developed a kind of can be fast, in a large number, efficiently, cheaply for the extracting method of wheat stripe rust RNA.Concrete steps are as follows:
1. in RNA Bechtop, take out the 2.0mL RNase free centrifuge tube that needs quantity, (cracking extracting solution composition is: 0.02M Tris-HCl pH=8.0 to add 800 μ L cracking extracting solutions; 0.2MNaCl; 0.005M EDTA; 1%SDS), each 10 μ L of 1% beta-mercaptoethanol and 16%PVP, are placed in centrifuge tube on ice after mixing, standby.
2. take 5mg wheat stripe rust uredospore, be loaded in 1.5mL centrifuge tube, bomb tube wall, is placed in and manages at the end to all spores gently.The 0.01M Tris-HCl (pH=8.0) that adds 30 μ L in pipe, the standing 5min of ice bath helps damping fluid to infiltrate in spore.
3. in precooling mortar, add 20mL liquid nitrogen flash freezer mortar, by the quick-frozen in new liquid nitrogen of above-mentioned centrifuge tube, after liquid-nitrogen boiling stops, reversing centrifuge tube one is put on the table gently simultaneously, spore in pipe and damping fluid are the complete ice shape that sticks together to be peeled off at the end with pipe, pours in the mortar of precooling.Add liquid nitrogen firmly to grind fast after 2-3 time, smalls meal is scraped down, concentrate and be transferred in the centrifuge tube of the 2.0mL that cracking extracting solution is housed that early-stage preparations are good, after vortex 2min, the standing 10min of ice bath.
4. add extraction liquid, its main component have an appointment the 5M NaAc (pH=4.8) of 270 μ L and the phenol of 750 μ L: chloroform: primary isoamyl alcohol (25: 24: 1), the standing 10min of ice bath, 4 ℃, the centrifugal 15min of 12000rpm, get supernatant (approximately 1000 μ L) and proceed to the 2.0mL centrifuge tube without RNA, add the 5MNaAc (pH=4.8) of approximately 300 μ L and the chloroform of 700 μ L, the standing 8min of ice bath, 4 ℃, the centrifugal 15min of 12000rpm, get supernatant, proceed to the 2.0mL centrifuge tube of RNase free, add the 3M NaAc (pH=5.2) of approximately 120 μ L and the Virahol of 800 μ L, place 30min for-20 ℃, 4 ℃, the centrifugal 15min of 12000rpm, abandon supernatant.
5. 80% ethanol that adds 500 μ L, bomb tube wall helps washing precipitation gently, and 4 ℃, the centrifugal 10min of 12000rpm, abandons supernatant, adds the dehydrated alcohol of 500 μ L, and bomb tube wall helps washing precipitation gently, and 4 ℃, the centrifugal 10min of 12000rpm, abandons supernatant.
6. add the RNase free water 43.5 μ L that process through DEPC to dissolve RNA, then add 10 * DNase Buffer, the 1 μ L DNaseI of 5 μ L and the RNase enzyme inhibitors of 0.5 μ L, slightly mix rear 37 ℃ of water-bath 30min.
7. add the 3M NaAc (pH=5.2) of approximately 5 μ L and the Virahol of 165 μ L, precipitation 30min, 4 ℃, the centrifugal 15min of 12000rpm, abandons supernatant.
8. 80% ethanol that adds 500 μ L, bomb tube wall helps washing precipitation gently, and 4 ℃, the centrifugal 10min of 12000rpm, abandons supernatant, adds the dehydrated alcohol of 500 μ L, and 4 ℃, the centrifugal 10min of 12000rpm, abandons supernatant.Add the RNase free water 20 μ L that DEPC processes to dissolve RNA ,-80 ℃ save backup.
By abovementioned steps, just can extract accurate, easy, fast complete wheat stripe rust RNA.What below show is some results that correlation test of the present invention obtains.
Accompanying drawing explanation
Accompanying drawing 1: shift wheat stripe rust uredospore with different methods from centrifuge tube and mortar completely.Situation after figure A uredospore shifts from centrifuge tube, figure B uredospore residual condition in mortar.In two figure, from left to right represent successively diatomite and uredospore (1: 1), pure uredospore, quartz sand and uredospore (1: 1).
2: four kinds of methods of accompanying drawing are extracted the electrophorogram of the total RNA of wheat stripe rust uredospore.Swimming lane 1-4 is respectively pure uredospore, and quartz sand is processed, and diatomite is processed and talcum powder is processed.Getting the total RNA of 4 μ L mixes 1 μ L10 * Loading Buffer and carries out loading, 120 volts of voltages, 15 minutes.
Accompanying drawing 3: shift wheat stripe rust uredospore completely by Tris-HCl (pH=8.0) method from centrifuge tube and mortar.Uredospore transfer case from centrifuge tube shown in figure A.The left side is directly to shift uredospore to after in mortar, has uredospore to remain on tube wall; The right is to shift uredospore by Tris-HCl (pH=8.0) method, does not have spore residual on tube wall.Uredospore residual condition in mortar shown in figure B.After the left side is direct liquid nitrogen grinding uredospore, a large amount of uredospores stick in mortar; The right is with after Tris-HCl (pH=8.0) method liquid nitrogen grinding uredospore, residual without uredospore in mortar.Total RNA integrity electrophorogram shown in figure C, swimming lane 1 is pure uredospore, swimming lane 2 is Tris-HCl (pH=8.0) methods.Getting the total RNA of 4 μ L mixes 1 μ L10 * Loading Buffer and carries out loading, 120 volts of voltages, 15 minutes.
Accompanying drawing 4: shift wheat stripe rust uredospore completely by the method for Tris-HCl (pH=8.0) and DEPC water from centrifuge tube and mortar.Uredospore transfer case from centrifuge tube shown in figure A.Uredospore residual condition in mortar shown in figure B.From left to right Tris-HCl (pH=8.0) and DEPC water law successively.RNA integrity electrophorogram shown in figure C, swimming lane 1-3 represents respectively pure uredospore, Tris-HCl (pH=8.0) method and DEPC water law.Getting the total RNA of 4 μ L mixes 1 μ L10 * Loading Buffer and carries out loading, 120 volts of voltages, 15 minutes.
Accompanying drawing 5: extract after wheat stripe rust RNA with different methods, uredospore is residual condition in mortar.Figure A is from left to right pure uredospore successively, and Tris-HCl (pH=8.0) and Bizol water law are extracted RNA.RNA integrity electrophorogram shown in figure B, swimming lane 1-3 is respectively pure uredospore, Tris-HCl (pH=8.0) method and Bizol water law.Getting the total RNA of 4 μ L mixes 1 μ L10 * Loading Buffer and carries out loading, 120 volts of voltages, 15 minutes.
Accompanying drawing 6: extract wheat stripe rust uredospore RNA with three kinds of different RNA lysates.A, B, C, D and E represent respectively to extract 1,5,10,20 and the uredinial electrophorogram of 30mg wheat stripe rust.F represents the residual condition (from left to right successively: LiCl, SDS and NaCl method) of wheat stripe rust in mortar after liquid nitrogen grinding.Swimming lane 1-3 is at A, B, and C, representative LiCl respectively in tetra-figure of D, SDS and NaCl method are extracted RNA.
Accompanying drawing 7: relatively three kinds of methods (LiCl, SDS and NaCl) are extracted the total RNA of wheat stripe rust.Solid line represents the total RNA after purifying, the RNA that dotted line representative is slightly carried.
Accompanying drawing 8: extract the total RNA of 5mg wheat stripe rust uredospore by three kinds of lysate methods, and carry out regular-PCR and electrophoresis detection with the elongation factor EF-1 of wheat stripe rust after its reverse transcription is become to cDNA, product size is 159bp.Swimming lane 1-4 represents respectively negative control, LiCl, SDS, NaCl method.
Test the complete transfer method research of 1 wheat stripe rust uredospore
Method explanation:
Wheat stripe rust volume is little, and quality is light, when weighing transfer, easily disperses and adheres to centrifugal tube wall, easily splashes and adheres to mortar bottom, thereby cause a large amount of RNA losses in liquid nitrogen grinding.This experiment is by relatively adding different solid matters (quartz sand, talcum powder and diatomite) and different liqs damping fluid (Tris-HCl, pH=8.0; Bizol and DEPC water) help the good and bad situation that uredospore shifts, determine that the optimum complete transfer method of wheat stripe rust uredospore is Tris-HCl (pH=8.0) method.Concrete operations are as follows:
The different solids treatment methods of table 1 shift wheat stripe rust uredospore
Table 2 different solutions treatment process shifts wheat stripe rust uredospore
The comparison of total RNA after four kinds of methods extraction wheat stripe rust uredospore purifying of table 3
Presentation of results:
1. solids treatment method shifts wheat stripe rust uredospore
As shown in accompanying drawing 1A, what diatomite and talcum powder (not providing) all can be very clean elutes wheat stripe rust uredospore.Quartz sand, because particle is large compared with uredospore, so a lot of uredospore is missed, is still bonded on tube wall.With after liquid nitrogen grinding, in the mortar that diatomite, talcum powder and quartz sand are processed, do not have uredospore residual, but the uredospore not dealing with have much stick to mortar bottom cannot scraping (accompanying drawing 1B).
Relatively total RNA (table 3) of four kinds of method extraction wheat stripe rust uredospore purifying, uses the RNA nucleic acid concentration of quartz sand and diatomite extraction higher than pure uredospore.After using talcum powder, uredinial RNA does not extract.Talcum powder main component is silicon-dioxide (60%) and magnesium oxide (30%), and the magnesium oxide in talcum powder has participated in RNA leaching process, has destroyed RNA extraction.The RNA concentration that diatomite extracts is far longer than pure uredospore and quartz sand method.
By electrophorogram, show (accompanying drawing 2), pure uredospore has complete RNA band, and the RNA degraded of extracting with quartz sand is serious.Seldom, degraded is serious, illustrates that RNA is degraded into more small shreds section for the RNA that electrophoresis showed is extracted although diatomite method detects very high nucleic acid concentration value.Talcum powder extracts RNA.
Evaluate the performance of three kinds of materials in leaching process, these material main components are silicon-dioxide (> 60%), and chemical property torpescence, does not react with organic reagent.Meanwhile, they have good homogeneity, can evenly mix with uredospore, and the uredospore on tube wall can be separated, and reduce the quantitative loss of uredospore.Talcum powder is finer and smoother, and energy and uredospore mix preferably, are difficult for being stained with wall.Diatomite is the finest and the smoothest, and uredospore mix best, can be stained with hardly wall (accompanying drawing 1A), in the time of in proceeding to liquid nitrogen, become a bulk, do not have and splash.Quartz sand, due to large mix with uredospore bad of its particle, attach a lot (accompanying drawing 1A), but in grinding, quartz sand also can be ground into powdery on tube wall, can help uredospore to grind fully.After liquid nitrogen grinding, when shifting in centrifuge tube, pure uredospore has is much stained with mortar bottom, and very difficult quilt scrapes off (accompanying drawing 1B), so lose more.Add quartz sand and talcous mortar when scraping mixture all than being easier to, and scrape and obtain very clean (accompanying drawing 1B).But, by the most direct electrophorogram, showing (accompanying drawing 2), the RNA that these three kinds of materials extract all has serious degraded, illustrates that other compositions in these three kinds of materials participate in RNA leaching process, thereby has destroyed the integrity of RNA.Finally, these three kinds of solid matters are not suitable for shifting in the uredinial process of wheat stripe rust.
2. liquid treating method shifts wheat stripe rust uredospore
Wheat stripe rust uredospore is transferred to liquid nitrogen and directly ground from centrifuge tube, on centrifuge tube tube wall, adhere to a lot of uredospores clean (accompanying drawing 3A is left), and after liquid nitrogen grinding, the bottom that uredospore is easy to stick to mortar is difficult for scraping (the accompanying drawing 3B left side).Utilize Tris-HCl (pH=8.0) method, in centrifuge tube, add 30 μ L Tris-HCl (pH=8.0), at standing 5min on ice, help damping fluid to infiltrate in spore.After liquid nitrogen flash freezer centrifuge tube, by centrifuge tube reversing and one put gently on the table, the spore in pipe and damping fluid are the complete ice shape that sticks together and peel off (accompanying drawing 3A is right) with the pipe end.Proceeded in mortar after liquid nitrogen grinding, uredospore does not all have on mortar and pestle residual (accompanying drawing 3B is right).
In centrifuge tube, add different solution (Tris-HCl pH=8.0, DEPC water, Bizol solution) on ice after standing and liquid nitrogen flash freezer, all can freeze to become stick together shape completely transfer in mortar of ice from centrifuge tube.But the transfer effect of Tris-HCl (pH=8.0) and Bizol (not providing) is better than DEPC water (accompanying drawing 4A is left), may be that organic buffer liquid more easily helps solution to infiltrate and adheres to spore compared with mineral ion water, reach complete transfer from centrifuge tube.But the residual condition of spore in mortar from grinding, after the mortar of Tris-HCl (pH=8.0) and DEPC water treatment extracts than Bizol solution clean (accompanying drawing 4B and accompanying drawing 5A).From complete transfer spore degree, both are more excellent compared with other for Tris-HCl (pH=8.0) method.
By the detection comparison (table 4) of nucleic acid concentration, the RNA nucleic acid concentration extracting by Tris-HCl (pH=8.0) method is the highest, and the nucleic acid concentration of Bizol and DEPC water extraction is also all higher than pure uredospore.From OD
260/280see, the nucleic acid quality of four processing is all good, and all within claimed range (1.8-2.2), from OD
260/230see OD
260/230> 2.0, and the removal of impurities that shows to desalt is more abundant.The demonstration of electrophorogram result, the RNA that Tris-HCl (pH=8.0) method is extracted has good integrity (Fig. 3 C), and the RNA of Bizol and DEPC water extraction is more complete (Fig. 4 C and Fig. 5 B) also.From RNA integrity and nucleic acid concentration, Tris-HCl (pH=8.0) method is more superior.
The comparison of total RNA after four kinds of methods extraction wheat stripe rust uredospore purifying of table 4
* the value in table is to repeat the mean value of experiment for three times.Remarkable with different letter representations in same hurdle
Property difference (P < 0.05=.
Test the comparison that 2 three kinds of lysates extract wheat stripe rust uredospore RNA
Method explanation:
Wheat stripe rust RNA extracts the methods that adopt test kit more, but cost is very high, is not suitable for the extraction of field great amount of samples RNA.This experiment is by relatively and optimize three kinds of common RNA lysates, developed a kind of can be fast, in a large number, efficiently, cheaply for the extraction lysate of wheat stripe rust RNA, specifically process in Table 5.
Three kinds of RNA lysate comparison of ingredients of table 5
Presentation of results:
Three kinds of lysates all can extract the uredinial complete total RNA of different mass wheat stripe rust (accompanying drawing 6).Wherein, in extracting 5mg uredospore, three kinds of nucleic acid concentrations different (table 6) that lysate extracts, are respectively 126.98ng/ μ L (LiCl), 333.10ng/ μ L (SDS) and 253.17ng/ μ L (NaCl).
(during < 15mg=uredospore, nucleic acid concentration is higher than NaCl and LiCl method extracting little quality for SDS method.On the contrary, NaCl method is when extracting large quality (> 15mg) wheat stripe rust uredospore, and its nucleic acid concentration is higher than LiCl and SDS method, and is and increases progressively trend (accompanying drawing 7).LiCl method is when extracting large quality uredospore, and its nucleic acid concentration approaches NaCl method, but when extracting little quality spore, its nucleic acid quality is lower than NaCl method.
By comparing slope, find, the slope of NaCl method is R to the maximum
2=0.999, LiCl is R
2=0.996, and SDS is R
2=0.926.
Whether the RNA that detects three kinds of lysates extractions can be used in subsequent experimental.According to TaKaRa reverse transcription test kit specification sheets, total RNA reverse transcription is become to cDNA, and take it as template, with wheat stripe rust elongation factor EF-1, be that primer carries out regular-PCR detection, find all can amplify object band after RNA reverse transcription that three kinds of lysates extract, for subsequent experimental (accompanying drawing 8).
By above-mentioned, relatively show, NaCl method has more advantage at extraction large quality materials, especially wheat leaf blade (about 100mg) aspect.
Three kinds of lysates of table 6 extract the comparison of different mass wheat stripe rust uredospore RNA
Claims (6)
1. a wheat stripe rust RNA extracting method rapidly and efficiently, comprise test materials pre-treatment, cracking, RNA extraction, RNA precipitation and washing precipitation step, it is characterized in that, the 0.01M Tris-HCl preprocessing solution in early stage that has adopted pH8.0 in test materials preprocessing process, it can help to grind and shift completely wheat stripe rust.
2. extracting method claimed in claim 1, the cracking extracting solution wherein adopting in cleavage step comprises following composition: 0.02M Tris-HCl pH=8.0,0.2M NaCl, 0.005M EDTA, 1%SDS (W: V).
3. extracting method claimed in claim 2, wherein cracking extracting liq also comprises the composition of anti-oxidation, 1% beta-mercaptoethanol (V: V) and 16%PVP (W: V).
4. according to the extracting method described in claim 1-3 any one, wherein said method is specific as follows: (1) test materials shifts and the pre-treatment of anti-oxidation completely: take 5mg wheat stripe rust uredospore, be loaded in 1.5mL centrifuge tube, bomb tube wall gently, to all spores, be placed in and manage at the end, in pipe, add 30 μ L pretreatment fluids in early stage, the standing 5min of ice bath, in precooling mortar, add about 30mL liquid nitrogen flash freezer mortar, simultaneously by the quick-frozen in new liquid nitrogen of above-mentioned centrifuge tube, after liquid-nitrogen boiling stops, spore in pipe is transferred in the mortar of Liquid nitrogen precooler, firmly grind 2-3 time fast, the liquid nitrogen that at every turn adds 20-30mL, afterwards, smalls meal is scraped down, be transferred in the centrifuge tube of the 2.0mL that 800 μ L cracking extracting solutions are housed that early-stage preparations are good, on vortex instrument after vortex 2min, the standing 10min of ice bath, (2) extraction, remove impurity: add extraction liquid, after putting upside down and mixing, the static 10min of ice bath, 4 ℃, the centrifugal 15min of 12000rpm, gets supernatant, add the pH4.85M NaAc of 300 μ L and the chloroform of 700 μ L, put upside down and mix, the static 8min of ice bath, 4 ℃, the centrifugal 15min of 12000rpm, gets supernatant, (3) RNA precipitation: add conventional RNA precipitated liquid, place 30min, 4 ℃ for-20 ℃, the centrifugal 15min of 12000rpm, abandons supernatant, adds the 80% washing with alcohol precipitation of 500 μ L, 4 ℃, the centrifugal 10min of 12000rpm, abandons supernatant, adds the absolute ethanol washing precipitation of 500 μ L, 4 ℃, the centrifugal 10min of 12000rpm, abandons supernatant, obtains wheat stripe rust RNA.
5. extracting method according to claim 4,5M NaAc and phenol that wherein extraction liquid comprises following composition: pH=4.8: chloroform: primary isoamyl alcohol=25: 24: 1.
6. according to the extracting method described in claim 1-5 any one, the RNA that the method is applicable to wheat stripe rust uredospore, mycelium, haustorium, winter spore extracts.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108192892A (en) * | 2018-03-01 | 2018-06-22 | 杭州比格飞序生物科技有限公司 | A kind of kit and extracting method of hydroxyl nanometer magnetic bead method extraction RNA |
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Non-Patent Citations (3)
| Title |
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| NEWTON，ET AL: "Variation for isozymes and double-stranded RNA among isolates of Puccinia striiformis and two other cereal rusts", 《PLANT PATHOLOGY》 * |
| 余宇等: "条锈菌诱导下的小麦叶片总RNA 提取方法的比较及LD-PCR 扩增", 《麦类作物学报》 * |
| 张韧等: "虹豆单抱锈菌中病毒样颗粒中双链R N A 的存在", 《真菌学报》 * |
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|---|---|---|---|---|
| CN108192892A (en) * | 2018-03-01 | 2018-06-22 | 杭州比格飞序生物科技有限公司 | A kind of kit and extracting method of hydroxyl nanometer magnetic bead method extraction RNA |
| CN108192892B (en) * | 2018-03-01 | 2021-12-28 | 杭州比格飞序生物科技有限公司 | Kit for extracting RNA by hydroxyl nano-magnetic bead method and extraction method |
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