CN109652407A - A kind of extracting method of microalgae cell total serum IgE - Google Patents
A kind of extracting method of microalgae cell total serum IgE Download PDFInfo
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- CN109652407A CN109652407A CN201910099483.8A CN201910099483A CN109652407A CN 109652407 A CN109652407 A CN 109652407A CN 201910099483 A CN201910099483 A CN 201910099483A CN 109652407 A CN109652407 A CN 109652407A
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Abstract
The present invention discloses a kind of extracting method of microalgae cell total serum IgE.The present invention assists grinding in liquid by the way that microalgae to be placed in plant RNA extraction containing quartz sand, realizes a step grinding of microalgae is rapidly completed and handle with polysaccharide is removed, and the demand to microalgae biomass substantially reduces, and enormously simplifies experimental implementation, reduces experiment time-consuming.The present invention combines the broken of microalgae cell wall with the removal of polysaccharide, breaking-wall cell can be completed in 5min and polysaccharide removes, pass through the innovation and improvement of whole extracting method, so that microalgae RNA extraction time foreshortens within 30min, on this basis, all operations for realizing microalgae RNA extraction can carry out in common laboratory environment, without removing the special extraction environment of RNA enzyme.Experimental article also goes RNA enzyme to handle without DEPC water.Damage of the DEPC water to researcher's health is eliminated while mitigating preliminary preparation, protects the safety of experimenter.
Description
Technical field
The invention belongs to RNA to extract field, and in particular to a kind of extracting method of microalgae cell total serum IgE.
Background technique
Microalgae is that one kind contains chlorophyll, can be carried out the general name of a kind of tiny organism of photosynthesis autophyting growth.Due to
Its cell is rich in the nutriments such as polysaccharides, protein, fatty acid, and it has can use photosynthesis fast-growth etc.
Feature and get more and more people's extensive concerning, had broad application prospects in fields such as food, health care, medical treatment.Microalgae also due to
Its photosynthetic efficiency is high, growth cycle is short, fat content is high and the physics of microalgae biodiesel and chemical characteristic and conventional diesel
The features such as similar, becomes third generation biodiesel, as being most hopeful to realize industrialized biodiesel now.
Although microalgae has very high value and good application prospect, it realizes that industrialized product is seldom,
One of them critically important reason is the weakness of its basic research.Functional gene relative fluorescence quantitative analysis based on microalgae RNA,
Transcript profile sequencing and analysis play very important effect in the basis of microalgae and forward position research.And what microalgae RNA was extracted
Convenience also directly influences the tempo of integral experiment work.But about the extraction of microalgae RNA, there are still several at present
Problem:
Sample size demand is big when 1, microalgae RNA are extracted: it is usually liquid nitrogen grinding that traditional microalgae RNA, which extracts the first step, institute
With biggish sample size, so that dynamic monitoring microalgae rna level becomes difficult.
2, microalgal polysaccharide seriously affects RNA extraction: rich in polysaccharides in frustule, and polysaccharide can combine after microalgae wall breaking
RNA coprecipitation, so that the extraction quality degradation of RNA, and the content of microdisk electrode to its polysaccharide of later period increases, more
Increase the difficulty of RNA extraction.
The harm of 3, DEPC water: when general extraction RNA, all experimental articles, experiment, which produces, to go by DEPC water
RNA enzyme processing, and DEPC water be it is carcinogenic, seriously affect the personal safety of researcher.
Time-consuming for 4, microalgae RNA extraction: all experimental articles, workbench due to extracting microalgae RNA will be through past RNA enzyme
Processing, microalgae want liquid nitrogen grinding, and Trizol extracts the influence of the factors such as the basic time-consuming of RNA, so that general microalgae RNA is extracted
Time-consuming for method, on the one hand will affect the yield and integrality of RNA, is on the other hand less useful for the smooth development of integral experiment.
Therefore, study it is a kind of safely, effectively, quick microalgae cell method for extracting total RNA, have emphatically to the research of microalgae
The meaning wanted.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide a kind of microalgae cell total serum IgEs
Extracting method.This method has the characteristics that safely, effectively, quickly.
The purpose of the invention is achieved by the following technical solution:
A kind of extracting method of microalgae cell total serum IgE, includes the following steps:
(1) microalgae cell that 2~6mL algae solution centrifugal concentrating is collected is placed in 2mL centrifuge tube；0.5~1.0mL is added to plant
Object RNA extracts auxiliary liquid and 0.3~0.8g quartz sand, is centrifuged after vibrating smudge cells, collects supernatant, is used for subsequent RNA
It extracts；
Further include following steps:
(2) supernatant is placed in 1.5mL centrifuge tube, 0.5mL Trizol is added and mixes, 200 μ L chloroforms are added, acutely
Oscillation, after solution is fully emulsified, centrifugation, supernatant is transferred in new 1.5mL centrifuge tube；
(3) the isometric isopropanol of addition, 0.8~1 μ L nucleic acid settling agent, after the centrifuge tube that turns upside down mixes well, room temperature
It stands, supernatant is abandoned in centrifugation；
(4) 75% ethyl alcohol is added, after the centrifuge tube that turns upside down mixes well, supernatant is abandoned in centrifugation, and drying at room temperature is added 20
The RNase-free water of~30 μ L dissolves precipitating, obtains total serum IgE.
Preferably, the microalgae include Chlorella sacchrarophila, Chlorella pyrenoidosa,
In Scenedesmus obliquus, Chlamydomonas reinhardtii and Chlorella luteorividis extremely
Few one kind, but the application of this method is not limited to these microalgaes, is suitable for all microalgaes.
Preferably, the volume of algae solution described in step (1) is 4mL.
Preferably, in step (1), 0.5mL plant RNA extraction auxiliary liquid and 0.5g quartz sand is added.
Preferably, the revolving speed of centrifugal concentrating described in step (1) is 10000~12000rpm；More preferably
12000rpm。
Preferably, the auxiliary of plant RNA extraction described in step (1) liquid is the Fruit-mate of TaKaRa companyTM。
Preferably, the size of quartz sand described in step (1) is 5~20 mesh；More preferably 10 mesh.
Preferably, the time of oscillation described in step (1) is 3~8min；More preferably 5min.
Preferably, the condition being centrifuged after oscillation smudge cells described in step (1) is 10000~12000rpm centrifugation
1min；More preferably 12000rpm is centrifuged 1min.
Preferably, the condition of centrifugation described in step (2) is that 10000~12000rpm is centrifuged 1min；More preferably
12000rpm is centrifuged 1min.
Preferably, the dosage of nucleic acid settling agent described in step (3) is 1 μ L.
Preferably, nucleic acid settling agent described in step (3) is glycogen (Glycogen, 20mg/mL).
Preferably, the time being stored at room temperature described in step (3) is 8~10 minutes；More preferably 10 minutes.
Preferably, the condition of centrifugation described in step (3) is that 10000~12000rpm is centrifuged 2min；More preferably
12000rpm is centrifuged 2min.
Preferably, the dosage of 75% ethyl alcohol described in step (4) is 0.8~1mL；More preferably 1mL.
Preferably, the condition of centrifugation described in step (4) is that 10000~12000rpm is centrifuged 1min；More preferably
12000rpm is centrifuged 1min.
Preferably, the time of drying at room temperature described in step (4) is 2~5min；More preferably 2min.
All operations can carry out in common laboratory environment, without removing the special extraction environment of RNA enzyme, it is used from
The experimental articles such as heart pipe, pipette tips are not necessarily to DEPC and RNA enzyme are gone to handle.
The present invention has the following advantages and effects with respect to the prior art:
(1) present invention carries out oscillation grinding to microalgae by introducing quartz sand, instead of the liquid nitrogen grinding of microalgae, so that micro-
Demand when algae RNA is extracted to microalgae biomass substantially reduces, and simplifies experimental implementation, and it is time-consuming to reduce grinding.
(2) present invention assists grinding in liquid by the way that microalgae to be placed in the plant RNA extraction containing quartz sand, realizes
The grinding that microalgae is rapidly completed in one step is handled with polysaccharide is removed, and enormously simplifies experimental implementation, and it is time-consuming to reduce experiment.
(3) present invention promotes microalgae RNA also to shorten extraction time while extracting quality, eliminates traditional extraction process
Harm of the middle DEPC water to human body, has great significance to the research of microalgae.
(4) present invention combines the broken of microalgae cell wall with the removal of polysaccharide, and breaking-wall cell can be completed in 5min
It is removed with polysaccharide, by the innovation and improvement of whole extracting method, so that microalgae RNA extraction time foreshortens within 30min,
On the basis of this, all operations for realizing microalgae RNA extraction can carry out in common laboratory environment, without removing RNA enzyme
Special extraction environment.The experimental articles such as centrifuge tube used in experiment, pipette tips are also not necessarily to DEPC (diethyl
Pyrocarbonate, pyrocarbonic acid diethyl ester) water goes RNA enzyme to handle.DEPC water is eliminated while mitigating preliminary preparation
Damage to researcher's health protects the safety of experimenter.
Detailed description of the invention
Fig. 1 is Chlorella sacchrarophila FACHB-4 difference RNA extraction method result；Wherein, 1: this hair
Bright method (experimental group 1)；2: general T rizol method (experimental group 2)；3: the method for the present invention, but experimental article used is gone through DEPC water
RNA enzyme processing, proposes the place RNA (experimental group 3) using special.
Fig. 2 is the RNA result that different microalgae is extracted using new method；Wherein, 1:Chlorella
LuteorividisFACHB-1 (embodiment two)；2:Chlorella pyrenoidosa FACHB-5 (embodiment three)；3:
Chlamydomonas reinhardtii FACHB-265 (example IV)；4:Scenedesmus obliquusFACHB-416
(embodiment five).
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.Following embodiments be it is illustrative, be not restrictive, protection model of the invention cannot be limited with following embodiments
It encloses.
Embodiment one:
Experimental group 1 is that the chlorella Chlorella of culture for 24 hours is extracted with the method for the present invention
SacchrarophilaFACHB-4 (buys from Wuhan Chinese Academy of Sciences wildlife Germplasm Bank-fresh water algae library) RNA, step
It suddenly is 12000rpm centrifugal concentrating 4mL algae solution into 2mL screw socket centrifuge tube；0.5mL plant RNA extraction is added and assists liquid
(the Fruit-mate of TaKaRa companyTM), 0.5g quartz sand (10 mesh or so) shaker is added and vibrates 5min, 12000rpm, from
Heart 1min draws supernatant into new 1.5mL centrifuge tube；0.5mL Trizol is added, mixes, 200 μ L chloroforms are added, acutely shake
It swings, after solution is fully emulsified, 12000rpm is centrifuged 1min, and Aspirate supernatant is transferred in another new 1.5mL centrifuge tube；
Isometric isopropanol is added, 1 μ L glycogen (nucleic acid settling agent, Glycogen, 20mg/mL), the centrifuge tube that turns upside down mixes well
Afterwards, it is being stored at room temperature 10 minutes, 12000rpm, is being centrifuged 2min, abandoning supernatant；75% ethyl alcohol of 1mL is added, turn upside down centrifuge tube
Rear 12000rpm is mixed well, 1min is centrifuged, abandons supernatant；Liquid in centrifuge tube is carefully blotted only with 10 μ L liquid-transfering guns, room temperature
Dry 2min, the RNase-free water that 20 μ L are added dissolve precipitating.
Experimental group 2 is that general T rizol proposes RNA method, the steps include: that 20mL algae solution is collected by centrifugation, liquid nitrogen is fully ground；
2mL Trizol Reagent is added, aspirates repeatedly, until without conglomeration cell in extract；Trizol mixed liquor is sucked and is centrifuged
Guan Zhong is centrifuged 10min under 4 DEG C, 12,000g revolving speeds；Supernatant is transferred in 2mL centrifuge tube, places 5min at room temperature；It is added
0.2mL chloroform shakes 15s with hand energetically, places 2~3min at room temperature, lamination can be observed；In 4 DEG C, 12,000g revolving speeds
Lower centrifugation 15min；Upper strata aqueous phase is shifted to 1.5mL centrifuge tube, 0.5mL isopropanol is added, mixes well, liquid change can be observed
Muddy or microprecipitation, places 15min at room temperature；10min is centrifuged under 4 DEG C, 12,000g revolving speeds；Supernatant is abandoned, 1mL is added
Dehydrated alcohol mixes washing precipitating, is centrifuged 10min under 4 DEG C, 12,000g revolving speeds；It is repeated once step 9；Supernatant is abandoned, is inverted
Dry 5~10min；The molten RNA of 30 μ L RNase-free water weight is added.
Experimental group 3 is using the method for the present invention, and experimental article goes RNA enzyme to handle through DEPC water, and is mentioned using special
The place RNA.
RNA is extracted after terminating through electrophoresis detection, as a result as shown in Figure 1, the method for the present invention is used to extract
Tri- band of C.sacchrarophila FACHB-4 (6S, 18S, 28S) clearly, illustrates that RNA extraction result is good, can satisfy
Subsequent experimental requirement.The RNA 6S band extracted using common liquid nitrogen grinding Trizol method is non-to be always on, but 18S and 28S are very light,
Illustrate that its RNA total amount is high, but it is very serious to degrade.In the result of DEPC water process, tri- band of RNA is all than in conventional environment
The result of extraction wants bright, but difference is not very big.
In summary result can be extracted within 30min it is found that using the method for the present invention and obtain microalgae RNA, and wherein
All operations can be carried out in common laboratory environment, without removing the special extraction environment of RNA enzyme.Used in experiment
The experimental articles such as centrifuge tube, pipette tips also go RNA enzyme to handle without DEPC water.It is eliminated while mitigating preliminary preparation
Damage of the DEPC water to researcher's health.
Embodiment two:
Extracting the chlorella Chlorella luteorividis FACHB-1 of culture for 24 hours with the method for the present invention, (purchase is certainly
Wuhan Chinese Academy of Sciences wildlife Germplasm Bank-fresh water algae library) RNA, specific steps are the same as embodiment one.Extract result such as
Fig. 2, three band (6S, 18S, 28S) clearly, illustrate that RNA extraction result is good, can satisfy subsequent experimental and want as the result is shown
It asks.
Embodiment three:
Extracting the chlorella Chlorella pyrenoidosa FACHB-5 of culture for 24 hours with the method for the present invention, (purchase is certainly military
Chinese Chinese Academy of Sciences wildlife Germplasm Bank-fresh water algae library) RNA, specific steps are the same as embodiment one.Result is extracted as schemed
2, three band (6S, 18S, 28S) clearly, illustrate that RNA extraction result is good, can satisfy subsequent experimental requirement as the result is shown.
Example IV:
The Chlamydomonas reinhardtii Chlamydomonas reinhardtiiFACHB-265 of culture for 24 hours is extracted with this patent method
(buying from Wuhan Chinese Academy of Sciences wildlife Germplasm Bank-fresh water algae library) RNA, specific steps are the same as embodiment one.It extracts
As a result such as Fig. 2, three band (6S, 18S, 28S) clearly, illustrate that RNA extraction result is good, can satisfy subsequent reality as the result is shown
Test requirement.
Embodiment five:
The scenedesmus obliquus Scenedesmus obliquus FACHB-416 (purchase of culture for 24 hours is extracted with the method for the present invention
From Wuhan Chinese Academy of Sciences wildlife Germplasm Bank-fresh water algae library) RNA, specific steps are the same as example one.Result is extracted as schemed
2, three band (6S, 18S, 28S) clearly, illustrate that RNA extraction result is good, can satisfy subsequent experimental requirement as the result is shown.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of extracting method of microalgae cell total serum IgE, which comprises the steps of:
(1) microalgae cell that 2~6mL algae solution centrifugal concentrating is collected is placed in 2mL centrifuge tube；0.5~1.0mL plant is added
RNA extracts auxiliary liquid and 0.3~0.8g quartz sand, is centrifuged after vibrating smudge cells, collects supernatant, mentions for subsequent RNA
It takes.
2. the extracting method of microalgae cell total serum IgE according to claim 1, it is characterised in that:
Further include following steps:
(2) supernatant is placed in 1.5mL centrifuge tube, 0.5mL Trizol is added and mixes, 200 μ L chloroforms are added, acutely vibrate,
After solution is fully emulsified, centrifugation, supernatant is transferred in new 1.5mL centrifuge tube；
(3) isometric isopropanol is added, 0.8~1 μ L nucleic acid settling agent is stored at room temperature after the centrifuge tube that turns upside down mixes well,
Supernatant is abandoned in centrifugation；
(4) 75% ethyl alcohol is added, after the centrifuge tube that turns upside down mixes well, supernatant is abandoned in centrifugation, and 20~30 μ are added in drying at room temperature
The RNase-free water of L dissolves precipitating, obtains total serum IgE.
3. the extracting method of microalgae cell total serum IgE according to claim 1 or 2, it is characterised in that:
The microalgae include but is not limited to Chlorella sacchrarophila, Chlorella pyrenoidosa,
In Scenedesmus obliquus, Chlamydomonas reinhardtii and Chlorella luteorividis extremely
Few one kind.
4. the extracting method of microalgae cell total serum IgE according to claim 2, it is characterised in that:
The volume of algae solution described in step (1) is 4mL；
In step (1), 0.5mL plant RNA extraction auxiliary liquid and 0.5g quartz sand is added；
The dosage of nucleic acid settling agent described in step (3) is 1 μ L.
5. the extracting method of microalgae cell total serum IgE according to claim 1 or 2, it is characterised in that:
The revolving speed of centrifugal concentrating described in step (1) is 10000~12000rpm；
The size of quartz sand described in step (1) is 5~20 mesh；
The time of oscillation described in step (1) is 3~8min；
The condition being centrifuged after oscillation smudge cells described in step (1) is that 10000~12000rpm is centrifuged 1min.
6. the extracting method of microalgae cell total serum IgE according to claim 5, it is characterised in that:
The revolving speed of centrifugal concentrating described in step (1) is 12000rpm；
The size of quartz sand described in step (1) is 10 mesh；
The time of oscillation described in step (1) is 5min；
The condition being centrifuged after oscillation smudge cells described in step (1) is that 12000rpm is centrifuged 1min.
7. the extracting method of microalgae cell total serum IgE according to claim 2, it is characterised in that:
The condition of centrifugation described in step (2) is that 10000~12000rpm is centrifuged 1min；
The time being stored at room temperature described in step (3) is 8~10 minutes；
The condition of centrifugation described in step (3) is that 10000~12000rpm is centrifuged 2min.
8. the extracting method of microalgae cell total serum IgE according to claim 7, it is characterised in that:
The condition of centrifugation described in step (2) is that 12000rpm is centrifuged 1min；
The time being stored at room temperature described in step (3) is 10 minutes；
The condition of centrifugation described in step (3) is that 12000rpm is centrifuged 2min.
9. the extracting method of microalgae cell total serum IgE according to claim 2, it is characterised in that:
The dosage of 75% ethyl alcohol described in step (4) is 0.8~1mL；
The condition of centrifugation described in step (4) is that 10000~12000rpm is centrifuged 1min；
The time of drying at room temperature described in step (4) is 2~5min.
10. the extracting method of microalgae cell total serum IgE according to claim 9, it is characterised in that:
The dosage of 75% ethyl alcohol described in step (4) is 1mL；
The condition of centrifugation described in step (4) is that 12000rpm is centrifuged 1min；
The time of drying at room temperature described in step (4) is 2min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111826373A (en) * | 2019-04-22 | 2020-10-27 | 中国科学院大连化学物理研究所 | Preparation method of microalgae total ribonucleic acid sample |
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| WO2015170280A1 (en) * | 2014-05-07 | 2015-11-12 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | Green microalgae lacking a functional dyrkp-1 gene, for use for increased production of feedstock |
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Application publication date: 20190419 |