CN102978196A - Method for extracting total RNA (Ribose Nucleic Acid) and total protein of rotifer - Google Patents
Method for extracting total RNA (Ribose Nucleic Acid) and total protein of rotifer Download PDFInfo
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- CN102978196A CN102978196A CN2012104989964A CN201210498996A CN102978196A CN 102978196 A CN102978196 A CN 102978196A CN 2012104989964 A CN2012104989964 A CN 2012104989964A CN 201210498996 A CN201210498996 A CN 201210498996A CN 102978196 A CN102978196 A CN 102978196A
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- wheel animalcule
- centrifuge tube
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- rotifer
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Abstract
The invention relates to a method for extracting total RNA (Ribose Nucleic Acid) and total protein of rotifer. According to the method, freshwater rotifer, general rotifer culture solution, food microalgae, culture solution of the food microalgae, a microscreen, an anatomical lens, a low-temperature refrigerator, a cell crusher, a refrigerated centrifuge, a charging pump, PVP (Polyvinyl Pyrrolidone), PBS (Phosphate Buffer Solution) and general RNA extracting agent (including TRIZOL, chloroform, isopropanol, DEPC (Diethylpyrocarbonate) water and ethanol) are utilized. The rotifer is cultured by individual cloning through artificial fluid medium; the food microalgae are freshwater nannochloris oculatas; and the pH (Potential of Hydrogen) of the PBS is 7.0. According to the method, the charging and continuous lighting method under constant temperature is adopted, so that a large number of rotifers can be quickly obtained, the microalgae can be removed, and living rotifers are enriched to accept the extraction of the total RNA and the total protein. By adopting the method, a large number of rotifers can be conveniently, quickly and economically collected, the rotifers can be used for molecular biology study, the influence due to microalgae can be effectively removed, and the rotifers are applicable to laboratory investigation.
Description
Technical field
The invention belongs to aquatic products subject biological technical field, specifically relate to a kind of method of extracting aquatic zooplankton rotifer total protein and total RNA.
Background technology
Wheel animalcule (rotifer) is Freshwater ecosystems major function group, and the early stage opening biological feed of 90% above fish also is environmental pollution and environmental risk assessment animal subject.The extraction of total protein and total RNA is the basis of carrying out molecular biology research.Because the wheel animalcule individuality is little, move about fast, the live body wheel animalcule that is difficult to the enrichment q.s carries out molecular biological research.The present invention has required equipment characteristics simple, easy and simple to handle.
Summary of the invention
The purpose of this invention is to provide a kind of method of extracting the total RNA of wheel animalcule and total protein.
Institute's supplying method comprises rotifer common nutrient solution preparation, the preparation of bait micro-algae nutrient solution, live body wheel animalcule beneficiation technologies, little algae removal method, total RNA method for releasing, ultrasonication method.
Wheel animalcule total protein of the present invention and method for extracting total RNA may further comprise the steps:
(1) wheel animalcule is cultivated;
(2) little algae is removed: the wheel animalcule nutrient solution is packed in the 50ml centrifuge tube, 4500rpm, 4 ℃ centrifugal 5 minutes, little algae sinks to the centrifuge tube bottom, carefully pours out the supernatant that contains wheel animalcule, this step can repeat 2-3 time until algae is all removed;
(3) wheel animalcule enrichment: 300 purpose screen clothes are converted into funnel-form, frame is on beaker, draw the wheel animalcule nutrient solution of removing little algae with glue head dropper, splash into slowly screen bottom, elimination moisture, with the distilled water flushing for several times, under anatomical lens, with the 10-100ml liquid-transfering gun wheel animalcule is drawn onto in the 1.5ml centrifuge tube afterwards;
(4) freezing: as under super clean bench, wheel animalcule to be transferred in the centrifuge tube without the RNA enzyme, added 1mlTRIZOL, turn upside down for several times, behind the mixing centrifuge tube is put into-20 ℃ of refrigerators, make it fully icing;
(5) total RNA extracts: the extracting solution room temperature that will add TRIZOL is melted, and 12000 turn 4 ℃ of centrifugal 5min; The careful supernatant of drawing moves in the new 1.5ml centrifuge tube, about 750ul, and the chloroform of adding 150ul (supernatant volume 1/5), with the violent concussion 15s of hand, fully emulsified rear room temperature leaves standstill 5min, and 12000 turn 4 ℃ of centrifugal 15min; Carefully from whizzer, take out, divide three layers of (colourless supernatant, middle layer white DNA, colored lower floor is organic phase protein), the careful supernatant of drawing moves in the new 1.5ml centrifuge tube, add isopyknic Virahol, prepare 75% ethanol [preparation of diethylpyrocarbonate (DEPC) water] behind the mixing that turns upside down during leaving standstill 10min(under 15-30 ℃, 12000 turn 4 ℃ of centrifugal 10min, abandon supernatant, slowly add 75% ethanol 1ml, turn upside down, 12000 turn 4 ℃ of centrifugal 5min, the careful ethanol of removing, drying at room temperature 2-5min adds 30ulDEPC water ,-70 ℃ of preservations.(above operation is all carried out in aseptic operating platform, and the centrifuge tube of using and rifle are first-class all without the RNA enzyme)
(6) cytoclasis: add in step 3 centrifuge tube 3ml1% polyvinylpyrrolidone (PVP K-30) (preparation of PBS phosphoric acid buffer, PH=7.0), concussion mixing, ultrasonication 10-15 time (power 300W, fragmentation time length 2S, interval 5S).Above all operations all carries out on ice.
(7) total protein extracts: 8000 turn after broken, 4 ℃ centrifugal 10 minutes, get supernatant.
Use substratum to be substratum commonly used among the present invention, wherein the wheel animalcule collective media is wheel animalcule human configuration substratum, and every liter contains NaHCO
36mg, CaSO
4.2H
2O 60mg, MgSO
4.7H
2O 123mg, KCl 4mg.Utilize as required HCl and NaOH to regulate pH value about 7.5.Bait micro-algae adopts the BBM substratum, and every liter contains NaNO
3250mg, CaCl
2.2H
2O MgSO
4.7H
2O 75mg, K
2HPO
475mg, KH
2PO
4175mg, NaCl 25 trace element and each 2mL of VITAMIN.
The 20-40W fluorescent lamp is adopted in illumination, and light application time 24 hours, temperature are controlled at 25-30 ℃, shake several times every day or utilize 10-15 rev/min of rotation culture apparatus rotating speed.
The said screen cloth of the present invention is 300 purpose filter screens, and the aperture is 54 microns, can elimination unnecessary moisture and remaining little algae, and (similar with the filter paper using method, Fig. 1) frame splashes into the wheel animalcule nutrient solution on beaker slowly during use it to be folded into funnel-form.
It is because wheel animalcule is too small that the present invention adopts freezing, and the common grinding rod can not be effectively with its fragmentation, and the cracking of wheel animalcule polypide meeting nature discharges RNA after the freeze thawing.
The present invention adopts the ultrasonication can effective broken wheel animalcule cell, discharges intracellular complexes, comprises total protein.
The concentration of the said polyvinylpyrrolidone of the present invention (PVP K-30) is 1%, and is now with the current by the PBS phosphoric acid buffer of PH=7.0.
Principle of the present invention is:
A. algae removal principle: wheel animalcule can move about fast, and centrifugal rear wheel animalcule can move about in supernatant liquor rapidly, thereby can effectively isolate little algae;
B. live body enrichment principle: the filtration of microscreen can be removed unnecessary moisture and remaining algae;
The c.RNA release principle: after adding TRIZOL, the freezing polypide of can breaking discharges RNA;
D. cytoclasis principle: ultrasonication can well destroy polypide, discharges intracellular protein.The method is easy and simple to handle, and fast, economy is convenient to use.
Description of drawings
Fig. 1 is that screen cloth uses synoptic diagram.
Fig. 2 is highdensity wheel animalcule after the enrichment.
Fig. 3 is comparison diagram before and after little algae is removed.
Embodiment
Employed term unless other explanation is arranged, generally has the implication that those of ordinary skills understand usually in the present invention.
Below in conjunction with specific embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following embodiment, various processes and the method do not described in detail are ordinary methods as known in the art.The source of agents useful for same, trade(brand)name and be necessary to list its moiety person are all indicated when occurring first, thereafter used identical reagent if no special instructions, all identical with the content of indicating first.
Embodiment 1: the removal of feed algae
(1) wheel animalcule is cultivated
Utilize No. 13 seston nets to fish for biopsy sample 200mL from the cultivating pool top layer.Take back the laboratory.Select 1 and carry non-mictic female, move in the micropore Tissue Culture Plate.Micropore adds Nannochloropsis oceanica concentration 3 * 10
6The wheel animalcule of ind/mL mixes suspension.Continuous illumination cultivation under the 20-40W fluorescent lamp, temperature 25-28 ° C, continuous charge are cultivated.Select 50 individualities that carry amictic egg after 2 days and move into 100mL Nannochloropsis oceanica concentration 3 * 10
6The wheel animalcule of ind/mL is cultivated suspension.
(2) algae removal
The wheel animalcule nutrient solution is packed in the 50ml centrifuge tube, 4500rpm, 4 ℃ centrifugal 5 minutes, little algae sinks to the centrifuge tube bottom, carefully pours out the supernatant that contains wheel animalcule, this step can repeat 2-3 time until algae is all removed.
Embodiment 2: the enrichment of live body wheel animalcule
300 purpose screen clothes are converted into funnel-form, and frame is (Fig. 1) on beaker, draws the wheel animalcule nutrient solution of removing little algae with glue head dropper, splash into slowly screen bottom, elimination moisture with the distilled water flushing for several times, is drawn onto wheel animalcule in the 1.5ml centrifuge tube with the 10-100ml liquid-transfering gun under anatomical lens afterwards.
Embodiment 3: total RNA extracts
Tradition is ground and the total RNA extraction efficiency of freezing fragmentation compares
Behind the algae removal, the wheel animalcule break process adopts following manner: process one: it is transferred in the preprepared grinding rod, add 1mlTRIZOL, grind for several times; Process two: wheel animalcule is transferred to without in the RNA enzyme centrifuge tube, added 1mlTRIZOL, put into cryogenic refrigerator freezing (about 30min), then take out on ice and melt: contrast: wheel animalcule is transferred to without after in the RNA enzyme centrifuge tube, adds 1mlTRIZOL, shakes up and down 10S.It is identical that RNA extracts the subsequent operations step.It is as shown in table 1 that RNA extracts the result, freezingly can extract efficiently the total RNA of wheel animalcule, and other operations all can reduce the RNA extraction efficiency.
Table 1 wheel animalcule total rna concentration pH-value determination pH
Embodiment 4: the extraction of total protein
Cytoclasis: add 3ml in step 3 centrifuge tube, massfraction is the preparation of 1%PVP K-30(PBS phosphoric acid buffer, PH=7.0), concussion mixing, ultrasonication 10-15 time (power 300W, fragmentation time length 2S, interval 5S).Above all operations all carries out on ice.
Total protein extracts: 8000 turn after broken, 4 ℃ centrifugal 10 minutes, get supernatant.
Determining of the broken number of times of best excusing from death:
The ultrasonication number of times is respectively 20 times, 15 times, and 10 times, 5 times.The final protein content that extracts is as shown in table 2, and broken number of times protein concentration in the time of 10-20 time is the highest, and the ultrasonic structure that can destroy albumen of crossing makes it lose its physiologically active, and ultrasonic number of times is very few, and the polypide fragmentation is insufficient, is unfavorable for the abundant release of albumen.
The broken number of times of the different excusing from death of table 2. is on the impact of total protein concentration
Claims (1)
1. the method extracted of the total RNA of wheel animalcule and total protein, its feature may further comprise the steps:
(1) wheel animalcule is cultivated
(2) little algae is removed: the wheel animalcule nutrient solution is packed in the 50ml centrifuge tube, 4500rpm, 4 ℃ centrifugal 5 minutes, little algae is deposited on the centrifuge tube bottom, pours out the supernatant that contains wheel animalcule, this step repeats 2-3 time until algae is all removed;
(3) wheel animalcule enrichment: 300 purpose screen clothes are converted into funnel-form, and frame is drawn the wheel animalcule nutrient solution of removing little algae with glue head dropper on beaker, splash into screen bottom, elimination moisture with the distilled water flushing for several times, is drawn onto wheel animalcule in the 1.5ml centrifuge tube with the 10-100ml liquid-transfering gun under anatomical lens afterwards;
(3) freezing: as under super clean bench, wheel animalcule to be transferred in the centrifuge tube without the RNA enzyme, added 1mlTRIZOL, turn upside down for several times, behind the mixing centrifuge tube is put into-80 ℃ of refrigerators, make it fully icing;
(4) total RNA extracts: the extracting solution room temperature that will add TRIZOL is melted, and 12000 turn 4 ℃ of centrifugal 5min; Draw supernatant and move in the new 1.5ml centrifuge tube, add chloroform, concuss 15s, fully emulsified rear room temperature leaves standstill 5min, and 12000 turn 4 ℃ of centrifugal 15min; From whizzer, take out, divide three layers in the centrifuge tube, draw supernatant and move in the new 1.5ml centrifuge tube, add isopyknic Virahol, leave standstill 10min behind the mixing that turns upside down under 15-30 ℃, 12000 turn 4 ℃ of centrifugal 10min, abandon supernatant, slowly add 75% ethanol 1ml, turn upside down, 12000 turn 4 ℃ of centrifugal 5min, remove ethanol, drying at room temperature 2-5min adds 26ulDEPC water ,-70 ℃ of preservations;
(5) cytoclasis: in step 3 centrifuge tube, add 3ml 1%PVP, concussion mixing, ultrasonication 10-20 time, power 300W, broken time length 2S, interval 5S; This step operation is all carried out on ice;
(6) total protein extracts: 4000 turn after broken, 4 ℃ centrifugal 10 minutes, get supernatant.
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| CN201210498996.4A CN102978196B (en) | 2012-11-29 | 2012-11-29 | Method for extracting total RNA (Ribose Nucleic Acid) and total protein of rotifer |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103882009A (en) * | 2014-03-19 | 2014-06-25 | 信阳市农业科学院 | Method for extracting total ribonucleic acid (RNA) of empoasca onukii matsuda imago |
| CN104094901A (en) * | 2014-07-11 | 2014-10-15 | 江苏农牧科技职业学院 | Indoor high-density rotifer culture harvest method applicable to molecular biology studies |
| CN105126443A (en) * | 2014-09-03 | 2015-12-09 | 安徽科技学院 | Filter paper filtering method in chemical experiment |
-
2012
- 2012-11-29 CN CN201210498996.4A patent/CN102978196B/en not_active Expired - Fee Related
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103882009A (en) * | 2014-03-19 | 2014-06-25 | 信阳市农业科学院 | Method for extracting total ribonucleic acid (RNA) of empoasca onukii matsuda imago |
| CN104094901A (en) * | 2014-07-11 | 2014-10-15 | 江苏农牧科技职业学院 | Indoor high-density rotifer culture harvest method applicable to molecular biology studies |
| CN104094901B (en) * | 2014-07-11 | 2016-07-06 | 江苏农牧科技职业学院 | A kind of indoor high-density wheel animalcule being suitable to molecular biology research cultivates collecting method |
| CN105126443A (en) * | 2014-09-03 | 2015-12-09 | 安徽科技学院 | Filter paper filtering method in chemical experiment |
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