CN103820433A - Method for extracting mRNA (messenger ribose nucleic acid) from tatty tissue - Google Patents
Method for extracting mRNA (messenger ribose nucleic acid) from tatty tissue Download PDFInfo
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- CN103820433A CN103820433A CN201410086548.2A CN201410086548A CN103820433A CN 103820433 A CN103820433 A CN 103820433A CN 201410086548 A CN201410086548 A CN 201410086548A CN 103820433 A CN103820433 A CN 103820433A
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Abstract
The invention discloses a method for extracting an mRNA (messenger ribose nucleic acid) from tatty tissue. The method comprises the following steps: S1, taking fatty tissue, and obtaining a total RNA solution with a Trizol method; and S2, adopting an RNA purification column to purify the total RNA solution so as to obtain the mRNA. An mRNA sample extracted by adopting the technical scheme of the invention is high in purity and good in integrity, can well meet requirements for high-throughput sequencing transcriptome database creation, and is low in time consumption and cost.
Description
Technical field
The present invention relates to technical field of molecular biology, in particular to a kind of method of extracting mRNA from the fatty tissue of richness.
Background technology
The extraction of total RNA is one of molecular biological substance, and high-quality RNA is the prerequisite of carrying out the researchs such as gene clone, genetic expression, gene function analysis.First RT-PCR, Real-Time PCR, Northern, cDNA library build carrying out of equimolecular biological study must obtain total RNA that purity is high, integrity is good.How the quality of total RNA is judged by Agilent 2100 chip biological analysers, this analyser has been proved to be alternative gel electrophoresis technology of wasting time and energy, rapid automatized high-quality numerised data can be provided, represent with the complete number of RNA (RIN).For animal tissues, in the time that reaching 7.0, RIN value could meet follow-up requirement for construction data base.Fatty tissue is an important endocrine organ, and its participates in the multiple Physiological and Biochemical Metabolism of body, the research of animal adipose tissue to fatty tissue grow, meat etc. is significant.Fatty tissue unit volume cell count is less, and the total RNA content in unit cell is relatively less (Mersmann, Harry J.Seminars in Plastic Srugery, 2002 also, 16 (2): 195～197), this has increased the difficulty of extracting total RNA from fatty tissue.
At present, the method for extracting fatty tissue has the method for Trizol and some test kits, but total RNA effect of directly extracting according to the operation of test kit specification sheets is unsatisfactory.So people improve test kit and Trizol method, after improving, can extract preferably total RNA(Wujiang dimension, Yang Gongshe, Sun Chao. the improvement [J] of Methods for Extracting Total RNA from Adipose. biotechnology circular, 2005,5:75-77,81).But in experimentation, find, can not meet the follow-up group requirement for construction data base of transcribing with the total RNA of Roll fat that above-mentioned improved Trizol method extracts.Therefore, need badly and develop a kind of method of extracting Roll fat mRNA to meet the follow-up group requirement for construction data base of transcribing.
Summary of the invention
The present invention aims to provide a kind of method of extracting mRNA from the fatty tissue of richness, can not meet the technical problem of transcribing group requirement for construction data base to solve the mRNA extracting in prior art from the fatty tissue of richness.
To achieve these goals, according to an aspect of the present invention, provide a kind of method of extracting mRNA from the fatty tissue of richness.The method comprises the following steps: S1, get rich fatty tissue, and adopt Trizol method to obtain total rna solution; S2, adopts RNA purification column to carry out purifying to total rna solution, obtains mRNA.
Further, Trizol method is that improved Trizol method is tieed up in Wujiang.
Further, the rich fatty Roll fat that is organized as.
Further, S1 comprises: Roll fat is carried out to homogenate, cracking, centrifugal removal grease, chloroform extracting, isopropanol precipitating, ethanol cleaning and resolution of precipitate, obtain total rna solution.
Further, S1 comprises: in liquid nitrogen, by Roll fat grind into powder, in Roll fat, add Trizol, and concussion mixes rear leaving standstill; Centrifugal removal grease, carries out after extracting the supernatant liquor obtaining with chloroform, precipitates with Virahol, and the precipitation obtaining is washed to rear use water dissolution with 75% ethanol, obtains total rna solution.
Further, S1 comprises: S11, gets the Roll fat of liquid nitrogen flash freezer, in liquid nitrogen by Roll fat grind into powder, get 100～150mg Roll fat and put into the first centrifuge tube that 1.5ml Trizol is housed, after Roll fat and Trizol concussion is mixed, at room temperature leave standstill 10min; S12, by the first centrifuge tube at 4 ℃, 12000rpm, centrifugal 10min, centrifugal rear removal upper strata grease, proceeds to the second centrifuge tube by lower floor's liquid, centrifugal 5min removes upper strata grease, and lower clear liquid is proceeded in the 3rd centrifuge tube; S13 adds 300 μ l chloroforms in the 3rd centrifuge tube, on vortex oscillation device, vibrates and mixes, and room temperature leaves standstill 3～5min, natural phase-splitting; Then by the 3rd centrifuge tube at 4 ℃, the centrifugal 10min of 12000rpm; Supernatant liquor is proceeded to the 4th centrifuge tube, adds and the isopyknic chloroform of supernatant liquor, on vortex oscillation device vibration mix, room temperature leaves standstill 3～5min, natural phase-splitting, then by the 4th centrifuge tube at 4 ℃, the centrifugal 10min of 12000rpm; S14, gets in supernatant liquor to the five centrifuge tubes in the 4th centrifuge tube, add with the 5th centrifuge tube in the isopyknic Virahol of supernatant liquor, mix to be placed in-80 ℃ and place 20min, then 4 ℃, the centrifugal 15min of 12000rpm; Remove supernatant liquor, be precipitated; S15, with volume percent be the precipitation that obtains in 75% ethanol cleaning step S14 2 times, centrifugal 2min, draws ethanol with rifle head, is placed in drying at room temperature 5～10min, with the thorough dissolution precipitation of 100 μ l DEPC treated water, obtains total rna solution.
Further, 75% ethanol is the ddH being processed by 7.5 parts by volume dehydrated alcohols and 2.5 parts by volume DEPC
2o is formulated.
Further, the ddH that DEPC processed
2o is for to add 99.9 parts by volume ddH by 0.1 parts by volume DEPC
2o mesoscale eddies mixes the water that spend the night (spending the night for usually said spending the night in biology, is generally 12～16 hours) obtains through autoclave sterilization more herein.
Further, S2 is for adopting a day root RNA purification kit to carry out purifying to total rna solution.
Further, S2 comprises: in total rna solution, add 350 μ l solution RK, mix; Add 250 μ l dehydrated alcohols, mix, gained solution is proceeded in adsorption column CR2 together with precipitation, centrifugal 30 seconds of 12,000rpm, discards the waste liquid in collection tube; In adsorption column CR2, add 500 μ l rinsing liquid RW, room temperature is placed after 2min, and 12,000rpm, centrifugal 30 seconds, abandons waste liquid, CR2 is put into collection tube, repeat rinsing once, 12, the centrifugal 5min of 000rpm, removes residual liquid, and adsorption column CR2 is proceeded in the 6th centrifuge tube, add RNase-Free water, room temperature is placed after 2min, and 12, the centrifugal 2min of 000rpm, then repeat wash-out once, obtain the mRNA of purifying.
Extract the total RNA effect that is rich in fatty tissue and can not meet high-flux sequence and transcribe the problem of group requirement for construction data base for Trizol method, the preliminary total RNA being rich in fatty tissue that extracts of improved Trizol method is tieed up in the Wujiang using in the present invention, can from the fatty tissue of richness, isolate total RNA fast, whole process can complete in 1h; And because contain RNase inhibitor and can guarantee the integrity of total RNA product; Total RNA that the method is extracted can reduce the pollution of albumen and DNA greatly.Then select day root RNA purification kit to continue purifying to extract the total RNA obtaining by aforesaid method, this test kit is to utilize centrifugal adsorbing column technology, mRNA and pellosil is efficient, the combination of single-minded ground under high salt condition, can be simultaneously to greatest extent except deproteinize, inorganic ion and many organic impuritys, make obtain mRNA sample purity high, integrity good, can meet well high-flux sequence and transcribe group requirement for construction data base, and consuming time short, and cost is low.
Accompanying drawing explanation
The Figure of description that forms the application's a part is used to provide a further understanding of the present invention, and schematic description and description of the present invention is used for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 shows by improved Trizol method and extracts the total RNA electrophoresis result obtaining, and wherein M represents Marker, is Trans2K Plus, sample Roll fat stoste loading 1 μ l;
Fig. 2 shows and extracts total RNA Agilent 2100 detected results that obtain by improved Trizol method;
Fig. 3 shows according to the method for the embodiment of the present invention and extracts the mRNA electrophoresis result obtaining, and wherein M represents Marker, is Trans2K Plus, sample Roll fat stoste loading 1 μ l.
Fig. 4 shows according to the method for the embodiment of the present invention and extracts mRNA Agilent 2100 detected results that obtain.
Embodiment
It should be noted that, in the situation that not conflicting, the feature in embodiment and embodiment in the application can combine mutually.Describe below with reference to the accompanying drawings and in conjunction with the embodiments the present invention in detail.
The problem of extracting the mRNA effect that is rich in fatty tissue and can not meet high-flux sequence and transcribe group requirement for construction data base for Trizol method, the invention provides a kind of method of extracting mRNA from the fatty tissue of richness.
According to the present invention, a kind of typical embodiment, provides a kind of method of extracting mRNA from the fatty tissue of richness.The method comprises the following steps: S1, get rich fatty tissue, and adopt Trizol method to obtain total rna solution; S2, adopts RNA purification column to carry out purifying to total rna solution, obtains mRNA.Application the method obtain mRNA sample purity high, integrity good, can meet well high-flux sequence and transcribe group requirement for construction data base, and operating process is simple, cost is low.
Preferably, Trizol method be Wujiang tie up improved Trizol method (Wujiang dimension, Yang Gongshe, Sun Chao. the improvement [J] of Methods for Extracting Total RNA from Adipose. biotechnology circular, 2005,5:75-77,81).
A kind of typical embodiment according to the present invention, the rich fatty Roll fat that is organized as.Described S1 comprises: Roll fat is carried out to homogenate, cracking, centrifugal removal grease, chloroform extracting, isopropanol precipitating, ethanol cleaning and resolution of precipitate, obtain total rna solution.Preferably, in liquid nitrogen, by Roll fat grind into powder, in Roll fat, add Trizol, and concussion mixes rear leaving standstill; Centrifugal removal grease, carries out after extracting the supernatant liquor obtaining with chloroform, precipitates with Virahol, and the precipitation obtaining is washed to rear use water dissolution with 75% ethanol, obtains total rna solution.
Further preferred, S1 comprises: S11, gets the Roll fat of liquid nitrogen flash freezer, in liquid nitrogen by Roll fat grind into powder, get 100～150mg Roll fat and put into the first centrifuge tube that 1.5ml Trizol is housed, after Roll fat and Trizol concussion is mixed, at room temperature leave standstill 10min; S12, by the first centrifuge tube at 4 ℃, 12000rpm, centrifugal 10min, centrifugal rear removal upper strata grease, proceeds to the second centrifuge tube by lower floor's liquid, centrifugal 5min removes upper strata grease, and lower clear liquid is proceeded in the 3rd centrifuge tube; S13 adds 300 μ l chloroforms in the 3rd centrifuge tube, on vortex oscillation device, vibrates and mixes, and room temperature leaves standstill 3～5min, natural phase-splitting; Then by the 3rd centrifuge tube at 4 ℃, the centrifugal 10min of 12000rpm; Supernatant liquor is proceeded to the 4th centrifuge tube, adds and the isopyknic chloroform of supernatant liquor, on vortex oscillation device vibration mix, room temperature leaves standstill 3～5min, natural phase-splitting, then by the 4th centrifuge tube at 4 ℃, the centrifugal 10min of 12000rpm; S14, gets in supernatant liquor to the five centrifuge tubes in the 4th centrifuge tube, add with the 5th centrifuge tube in the isopyknic Virahol of supernatant liquor, mix to be placed in-80 ℃ and place 20min, then 4 ℃, the centrifugal 15min of 12000rpm; Remove supernatant liquor, be precipitated; S15, with volume percent be the precipitation that obtains in 75% ethanol cleaning step S14 2 times, centrifugal 2min, with rifle head absorption ethanol, be placed in drying at room temperature 5～10min, with 100 μ l DEPC(Diethypyrocar-bonate, diethylpyrocarbonate) the thorough dissolution precipitation of treated water, obtain total rna solution.
A kind of typical embodiment according to the present invention, S2, for adopting a day root RNA purification kit to carry out purifying to total rna solution, is not limited to a day root RNA purification kit certainly.In the time adopting a day root RNA purification kit to carry out purifying to total rna solution, S2 comprises: in RNA solution, add 350 μ l solution RK, mix; Add 250 μ l dehydrated alcohols, mix, gained solution is proceeded in adsorption column CR2 together with precipitation, centrifugal 30 seconds of 12,000rpm, discards the waste liquid in collection tube; In adsorption column CR2, add 500 μ l rinsing liquid RW, room temperature is placed after 2min, and 12,000rpm, centrifugal 30 seconds, abandons waste liquid, CR2 is put into collection tube, repeat rinsing once, 12, the centrifugal 5min of 000rpm, removes residual liquid, and adsorption column CR2 is proceeded in the 6th centrifuge tube, add RNase-Free water, room temperature is placed after 2min, and 12, the centrifugal 2min of 000rpm, then repeat wash-out once, obtain the mRNA of purifying.
Wherein, be the ddH being processed by 7.5 parts by volume dehydrated alcohols and 2.5 parts by volume DEPC with the ethanol that volume percent is 75%
2o is formulated.The ddH that DEPC processed
2o is for to add 99.9 parts by volume ddH by 0.1 parts by volume DEPC
2o mesoscale eddies mixes the water that spends the night and obtain through autoclave sterilization.
Further illustrate beneficial effect of the present invention below in conjunction with embodiment.
In the present embodiment, Roll fat used is fresh, healthy material.
Implementation process is as follows:
(1) get the Roll fat sample 500mg of liquid nitrogen flash freezer, be ground into rapidly powder with liquid nitrogen, fast about 150mg sample is transferred in the centrifuge tube that 1.5ml Trizol is housed, upper and lower concuss 5min left and right at room temperature leaves standstill 10min after sample and solution mix completely;
(2) 4 ℃, 12000rpm, centrifugal 10min, a large amount of greases are contained on centrifugal rear upper strata, remove with suction nozzle, and lower floor's liquid is proceeded to one and newly manage, and then centrifugal 5min, remove upper strata grease, proceed to clearly in a new pipe lower;
(3) add 300 μ l chloroforms, cover tightly pipe lid, on vortex oscillation device, vibration mixes, and room temperature leaves standstill 3-5min, makes its natural phase-splitting; 4 ℃, the centrifugal 10min of 12000rpm; Supernatant is proceeded to one and newly manage, add equal-volume chloroform, on vortex oscillation device, vibration mixes, and room temperature leaves standstill 3-5min, makes its natural phase-splitting, and 4 ℃, the centrifugal 10min of 12000rpm;
(4) get supernatant in new centrifuge tube, add isopyknic Virahol, mix to be placed in-80 ℃ and place 20min, then 4 ℃, the centrifugal 15min of 12000rpm;
(5) with 75%(v/v) ethanol washing and precipitating 2 times, then empty from 2min, draw ethanol with rifle head, be placed in drying at room temperature 5-10min, thoroughly dissolve RNA with 100 μ l DEPC treated waters and precipitate;
(6) in RNA solution, add 350 μ l solution RK, fully mix.Add afterwards 250 μ l dehydrated alcohols, fully mix, immediately gained solution is proceeded in adsorption column CR2 together with precipitation, the centrifugal 30sec of 12,000rpm, discards the waste liquid in collection tube.In adsorption column CR2, add 500 μ l rinsing liquid RW, room temperature is placed after 2min, 12,000rpm, and centrifugal 30sec, abandons waste liquid, and CR2 is put into collection tube.Repeat rinsing once, the centrifugal 5min of 12,000rpm, removes residual liquid, adsorption column CR2 is proceeded in a new centrifuge tube, add appropriate RNase-Free water, room temperature is placed after 2min, and 12, the centrifugal 2min of 000rpm, repeats wash-out once afterwards again, obtains the mRNA after purifying.
Quality examination: detect through agarose gel electrophoresis, what use the Trizol method of improvement and method of the present invention the results are shown in Figure 1 and Fig. 3, as seen in Figure 1 by after the total RNA electrophoresis extracting through Trizol method, three bands are more clearly there are at this swimming lane, not traction and large stretch of light-emitting zone always, the integrity that shows total RNA is better, electrophoretic band on the swimming lane of the mRNA extracting through method provided by the invention on Fig. 3 is also not occur traction and a sheet of light-emitting zone, also shows that mRNA integrity is all fine.Visible, use the Trizol method of improvement and the integrity of the RNA that method of the present invention obtains all good.Use Agilent 2100 accurately to detect mRNA integrity and the results are shown in Figure 2 and Fig. 4, the result of Fig. 2 shows, use the Trizol method RIN value of improvement to only have 5.8, do not reach high-flux sequence and transcribe group requirement for construction data base (RIN value is minimum is 7.0), visible by Fig. 4, use method RIN value of the present invention to reach 8.0, this sample can meet well high-flux sequence and transcribe group requirement for construction data base.
Wherein, the parameter in Fig. 2 is as shown in table 1:
Table 1
Parameter in Fig. 4 is as shown in table 2:
Table 2
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. a method of extracting mRNA from the fatty tissue of richness, is characterized in that, comprises the following steps:
S1, gets the fatty tissue of described richness, adopts Trizol method to obtain total rna solution;
S2, adopts RNA purification column to carry out purifying to described total rna solution, obtains mRNA.
2. method according to claim 1, is characterized in that, described Trizol method is that improved Trizol method is tieed up in Wujiang.
3. method according to claim 1, is characterized in that, the fatty Roll fat that is organized as of described richness.
4. method according to claim 3, is characterized in that, described S1 comprises: described Roll fat is carried out to homogenate, cracking, centrifugal removal grease, chloroform extracting, isopropanol precipitating, ethanol cleaning and resolution of precipitate, obtain described total rna solution.
5. method according to claim 4, is characterized in that, described S1 comprises: in liquid nitrogen, by described Roll fat grind into powder, in described Roll fat, add Trizol, and concussion mixes rear leaving standstill; Centrifugal removal grease, carries out after extracting the supernatant liquor obtaining with chloroform, precipitates with Virahol, and the precipitation obtaining is washed to rear use water dissolution with 75% ethanol, obtains described total rna solution.
6. method according to claim 5, is characterized in that, described S1 comprises:
S11, get the described Roll fat of liquid nitrogen flash freezer, in liquid nitrogen, by described Roll fat grind into powder, get Roll fat described in 100～150mg and put into the first centrifuge tube that 1.5ml Trizol is housed, after described Roll fat and described Trizol concussion is mixed, at room temperature leave standstill 10min;
S12, by described the first centrifuge tube at 4 ℃, 12000rpm, centrifugal 10min, centrifugal rear removal upper strata grease, proceeds to the second centrifuge tube by lower floor's liquid, centrifugal 5min removes upper strata grease, and lower clear liquid is proceeded in the 3rd centrifuge tube;
S13 adds 300 μ l chloroforms in described the 3rd centrifuge tube, on vortex oscillation device, vibrates and mixes, and room temperature leaves standstill 3～5min, natural phase-splitting; Then by described the 3rd centrifuge tube at 4 ℃, the centrifugal 10min of 12000rpm; Supernatant liquor is proceeded to the 4th centrifuge tube, adds and the isopyknic chloroform of described supernatant liquor, on vortex oscillation device vibration mix, room temperature leaves standstill 3～5min, natural phase-splitting, then by described the 4th centrifuge tube at 4 ℃, the centrifugal 10min of 12000rpm;
S14, gets in supernatant liquor to the five centrifuge tubes in described the 4th centrifuge tube, add with described the 5th centrifuge tube in the isopyknic Virahol of supernatant liquor, mix to be placed in-80 ℃ and place 20min, then 4 ℃, the centrifugal 15min of 12000rpm; Remove supernatant liquor, be precipitated;
S15, is that 75% ethanol cleans the precipitation that obtains in described step S14 2 times by volume percent, and centrifugal 2min, draws ethanol with rifle head, is placed in drying at room temperature 5～10min, with the thorough dissolution precipitation of 100 μ l DEPC treated water, obtains described total rna solution.
7. method according to claim 6, is characterized in that, described 75% ethanol is the ddH being processed by 7.5 parts by volume dehydrated alcohols and 2.5 parts by volume DEPC
2o is formulated.
8. method according to claim 7, is characterized in that, the ddH that described DEPC processed
2o is for to add 99.9 parts by volume ddH by 0.1 parts by volume DEPC
2o mesoscale eddies mixes the water that spends the night and obtain through autoclave sterilization.
9. method according to claim 1, is characterized in that, described S2 is for adopting a day root RNA purification kit to carry out purifying to described total rna solution.
10. method according to claim 9, is characterized in that, described S2 comprises:
In described total rna solution, add 350 μ l solution RK, mix; Add 250 μ l dehydrated alcohols, mix, gained solution is proceeded in adsorption column CR2 together with precipitation, centrifugal 30 seconds of 12,000rpm, discards the waste liquid in collection tube; In adsorption column CR2, add 500 μ l rinsing liquid RW, room temperature is placed after 2min, and 12,000rpm, centrifugal 30 seconds, abandons waste liquid, CR2 is put into collection tube, repeat rinsing once, 12, the centrifugal 5min of 000rpm, removes residual liquid, and adsorption column CR2 is proceeded in the 6th centrifuge tube, add RNase-Free water, room temperature is placed after 2min, and 12, the centrifugal 2min of 000rpm, then repeat wash-out once, obtain the mRNA of purifying.
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Cited By (1)
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