CN1904044A - One step method ribonucleic acid extraction agent - Google Patents
One step method ribonucleic acid extraction agent Download PDFInfo
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- CN1904044A CN1904044A CN 200610029556 CN200610029556A CN1904044A CN 1904044 A CN1904044 A CN 1904044A CN 200610029556 CN200610029556 CN 200610029556 CN 200610029556 A CN200610029556 A CN 200610029556A CN 1904044 A CN1904044 A CN 1904044A
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- ribonucleic acid
- plastics
- extraction agent
- supernatant liquor
- diethylpyrocarbonate
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Abstract
The present invention relates to an one-step method ribonucleic acid extraction reagent EZRNA. Its formula includes (by 1 L total amount) guanidinium isothiocyanate 3-5 M, sodium citrate 18-25 M, sodium laurate (by wt%) 0.3-0.6%, 2-mercaptoethanol 0.6-1.2 M and balanced acidic phenol (by volume%) 40-55%. Besides, said invention also provides the concrete steps of the invented one-step method ribonucleic acid extraction process by using said reagent.
Description
Technical field
One step method ribonucleic acid extraction agent of the present invention relates to the reagent and the extraction process thereof of a kind of single stage method extracting ribonucleic acid (to call RNA in the following text).
Background technology
In order to separate fast complete RNA from cell, many methods have all been used strong denaturant and have been made lysis, dissolving and the endogenic RNA enzyme of sex change simultaneously, and this method is formally published an article by Han (1987) etc. and Chirgwin (1979) etc. and is obtained detailed description.Because the method for Han etc. will be with the guanidine thiocyanate dissolving RNA of continuous a spot of 5M, so comparatively loaded down with trivial details.This method is almost replaced by the single stage method of Chomczynski and Sacchi for this reason, and this method does not need ultracentrifugation, thereby many samples can be operated and raising speed, and the present invention is the RNA extraction agent of researching and developing voluntarily on this basis.Chang Yong RNA extraction agent has following several in the market: Trizol (Life Technologies), TRI reagent (Molecular ResearchCenter), RNA-Stat-60 (Tel-Test) etc.
Summary of the invention
The object of the invention is to provide the reagent of the quick extracting RNA of a kind of single stage method.
A further object of the present invention provides a kind of technology of utilizing above-mentioned extractant extracting RNA.
For reaching above-mentioned purpose, the invention provides a kind of one step method ribonucleic acid extraction agent, its prescription is in the 1L total amount, guanidinium isothiocyanate 3～5M wherein, Trisodium Citrate 18～25M, month water glass is 0.3～0.6% by weight percentage, 2 mercapto ethanol 0.6～1.2M, and the acid phenol of balance by volume per-cent counts 40～55%.
The technological process of extracting RNA is:
Tissue after milling in a, the liquid nitrogen supernatant liquor of leaving away;
B, adding one step method ribonucleic acid extraction agent aspirate mixing repeatedly;
C, adding glycogen, thermal agitation, homogenate;
D, with syringe suction, shear thymus nucleic acid;
E, adding chloroform and iso pentane alcohol mixture or chloroform, thermal agitation, centrifugal;
Add Virahol in f, the supernatant liquor, leave standstill, centrifugal, remove supernatant liquor;
G, precipitation are with the alcohol washing, and be centrifugal, removes supernatant liquor;
H, traditional vacuum drying, volatilization alcohol;
I, throw out diethylpyrocarbonate deionized water dissolving.
As find the throw out indissoluble, can be with described throw out at 68 ℃ of following incubations, or dissolve with 100% deionized formamide.
For preventing RNA degraded or contaminated, the preparatory technology of the method for extracting ribonucleic acid is:
1, the staff must wear gloves, wear masks during extracting RNA;
2, clean the inside and outside of liquid getting device with 70% ethanol of diethylpyrocarbonate (to call DEPC in the following text) preparation;
3, plastics use disposable sterilized plastics or carry out following processing:
A, inject deionized water in glass beaker, adding DEPC, to make its final concentration be 0.05%;
B, pending plastics are put into the container of a high-temperature sterilization, inject the diethylpyrocarbonate deionized water (to call DEPC-H in the following text
2O), all parts of plastics all are dipped in the solution, in Fume Hoods, handle under 37 ℃ or the room temperature and spend the night;
C, with DEPC-H
2O pours in the waste liquid bottle, and with aluminium foil sealing, the high temperature and high pressure steam sterilization is no less than 30 minutes with container that the plastics of handling are housed;
D, the plastics of sterilizing are put cleaning and are located standby 80～90 ℃ of baking dryings down;
4, glass and metal are no less than 3 hours 250 ℃ of bakings.
For more effective extracting RNA, when tissue mass is that 1～10mg or cell count are 10
2～10
4The time, RNA extraction agent add-on is 800 μ l; Must not draw intermediate layer material when removing supernatant liquor, avoid RNA to lose.
The RNA rapid extraction method that the present invention relates to is come lysing cell by the RNA extraction agent individual event lysate that comprises guanidinium isothiocyanate and phenol, adds chloroform and produces two-phase---organic phase and water.By extracting, RNA then stays the upper strata aqueous phase in organic phase for DNA and albumen, makes things convenient for the later stage operation.
Advantage of the present invention is: compared with the prior art, the same with the effect of existing extractant, but it is cheap, be 1/10 of imported product, do not need cryopreservation, greatly reduced production cost, broken the monopolization situation of external product on Chinese market, and in the process that obtains final product or pure rna, can not waste to some extent, in addition, the present invention can preserve 1 month by normal temperature.
Embodiment
Single stage method RNA extraction agent of the present invention, concrete prescription (in the 1L total amount) is as follows: guanidinium isothiocyanate 3～5M, and Trisodium Citrate 18～25M, a month water glass is 0.3～0.6% by weight percentage, 2 mercapto ethanol 0.6～1.2M, the acid phenol of balance by volume per-cent counts 40～55%.
Extraction steps is as follows in regular turn:
In liquid nitrogen, tissue is milled into powder, take advantage of liquid nitrogen be not evaporated completely as yet full-time, with powder transfer in aseptic 1.5ml centrifuge tube.Cell directly adds centrifuge tube after counting, with 5,000rpm (rev/min) room temperature is centrifugal, removes supernatant liquor.
Add the RNA extraction agent, every 100mg tissue or 5 * 10
6Individual cell adds the RNA extraction agent of 1.0ml, aspirates mixing repeatedly.
Add glycogen, making final concentration is 250 μ g/ml, thermal agitation or use homogenizer homogenate.With syringe suction, the step of shearing DNA is: use the 1ml syringe, 26-G number (6#) syringe needle suction homogenate 15～20 times be with the shearing genomic dna, then directly from syringe with sample transfer in aseptic 1.5ml centrifuge tube.
Add 200 μ l chloroform/primary isoamyl alcohol (24: 1) or chloroforms, thermal agitation mixing 30s, on the desk centrifuge with 12, the centrifugal 5min of 000rpm room temperature.
Supernatant liquor is carefully transferred in the centrifuge tube of aseptic 1.5ml, added isopyknic Virahol, place 5min under the room temperature.Whizzer is with 12, and the centrifugal 5min of 000rpm room temperature carefully removes supernatant liquor.
RNA precipitates with 70% alcohol washed twice, each alcohol consumption 700 μ l, and whizzer is with 12, and the centrifugal 2min of 000rpm room temperature siphons away supernatant liquor as far as possible up hill and dale.
Traditional vacuum, dry 3～5min, or be placed under the room temperature alcohol is volatilized fully.
Throw out is with 30～50 μ l DEPC-H
2The O dissolving.
As find to precipitate indissoluble, 68 ℃ of following incubation 10min.For pancreas, RNA enzyme content is very high in the tissue such as kidney, and precipitation is dissolved with 100% deionized formamide.
The preparatory technology of extracting RNA is as follows:
1, must wear gloves during extracting RNA, wear masks because of the RNA enzyme causes the RNA degraded for avoiding.
2, according to the requirement of liquid getting device manufacturers liquid getting device is handled, avoided polluting.Adopt generally speaking with 70% ethanol of DEPC preparation and clean the inside and outside of liquid getting device.
3, plastics use disposable sterilized plastics as far as possible.Indicated the plastics of RNase-free, used, needn't handle again usually as not breaking a seal.For homemade plastics, can use after handling in principle, treatment process is as follows:
A, inject deionized water in glass beaker, adding DEPC, to make its final concentration be 0.05%.
B, pending plastics are put into the container of a high-temperature sterilization, inject DEPC-H
2O is dipped in the solution all parts of plastics, in Fume Hoods, handles under 37 ℃ or room temperature and spends the night.
C, with DEPC-H
2O carefully pours in the waste liquid bottle, and DEPC-H will be housed
2The container of the plastics that O handled is with aluminium foil sealing, and the high temperature and high pressure steam sterilization is no less than 30min.
D, the plastics of sterilizing are put cleaning and are located standby 80～90 ℃ of baking dryings down.
4, glass and metal need 250 ℃ of bakings more than 3 hours.
Lime light in the extraction steps of the present invention:
1, when tissue mass be that 1～10mg or cell count are 10
2～10
4The time, RNA extraction agent add-on is 800 μ l, aspirates mixing repeatedly with the rifle head.
When 2, removing supernatant liquor, do not draw any intermediate layer material, pollute otherwise chromosomal DNA can occur.
3, add Virahol centrifugal after, when removing supernatant liquor, note avoiding the RNA precipitation to lose.
4, after the alcohol washing, when removing supernatant liquor, note avoiding the RNA precipitation to lose.
Claims (6)
1, a kind of one step method ribonucleic acid extraction agent, in 1 liter of total amount, its prescription is:
Guanidinium isothiocyanate 3～5M, Trisodium Citrate 18～25M, a month water glass is 0.3～0.6% by weight percentage, 2 mercapto ethanol 0.6～1.2M, the acid phenol of balance by volume per-cent counts 40～55%.
2, a kind of method of utilizing ribonucleic acid extraction agent extracting ribonucleic acid according to claim 1, the extraction process process is as follows:
Tissue after milling in a, the liquid nitrogen supernatant liquor of leaving away;
B, adding one step method ribonucleic acid extraction agent aspirate mixing repeatedly;
C, adding glycogen, thermal agitation, homogenate;
D, with syringe suction, shear thymus nucleic acid;
E, adding chloroform and iso pentane alcohol mixture or chloroform, thermal agitation, centrifugal;
Add Virahol in f, the supernatant liquor, leave standstill, centrifugal, remove supernatant liquor;
G, precipitation are with the alcohol washing, and be centrifugal, removes supernatant liquor;
H, traditional vacuum drying, volatilization alcohol;
I, throw out diethylpyrocarbonate deionized water dissolving.
3, method according to claim 2 is characterized in that: described throw out is 68 ℃ of following incubation dissolvings.
4, method according to claim 2 is characterized in that: described throw out dissolves with 100% deionized formamide.
5, a kind of preparatory technology of the method at the described extracting ribonucleic acid of claim 2:
(1), the staff must wear gloves, wear masks during extracting ribonucleic acid;
(2), clean the inside and outside of liquid getting device with 70% ethanol of diethylpyrocarbonate preparation;
(3), plastics use disposable sterilized plastics or carry out following processing:
A, inject deionized water in glass beaker, adding diethylpyrocarbonate, to make its final concentration be 0.05%;
B, pending plastics are put into the container of a high-temperature sterilization, inject the diethylpyrocarbonate deionized water, all parts of plastics all are dipped in the solution, in Fume Hoods, handle under 37 ℃ or the room temperature and spend the night;
C, the diethylpyrocarbonate deionized water is poured in the waste liquid bottle, with aluminium foil sealing, the high temperature and high pressure steam sterilization is no less than 30 minutes with container that the plastics of handling are housed;
D, the plastics of sterilizing are put cleaning and are located standby 80～90 ℃ of baking dryings down;
(4), glass and metal are no less than 3 hours 250 ℃ of bakings;
6, method according to claim 2 is characterized in that:
When tissue mass is that 1～10mg or cell count are 10
2～10
4The time, the ribonucleic acid extraction agent add-on is 800 μ l; Must not draw intermediate layer material when removing supernatant liquor, avoid Yeast Nucleic Acid to lose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610029556 CN1904044A (en) | 2006-07-31 | 2006-07-31 | One step method ribonucleic acid extraction agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610029556 CN1904044A (en) | 2006-07-31 | 2006-07-31 | One step method ribonucleic acid extraction agent |
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| CN1904044A true CN1904044A (en) | 2007-01-31 |
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| CN 200610029556 Pending CN1904044A (en) | 2006-07-31 | 2006-07-31 | One step method ribonucleic acid extraction agent |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102443580A (en) * | 2010-10-15 | 2012-05-09 | 华中农业大学 | Reagent composition for separating total RNA in plant or microorganism and preparation method thereof |
| WO2021245660A1 (en) * | 2020-05-31 | 2021-12-09 | Ofek Eshkolot Research And Development Ltd. | Compositon for sampling body fluids and secretions for detecting pathogenic agents nucleic acids and for disinfection |
-
2006
- 2006-07-31 CN CN 200610029556 patent/CN1904044A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102443580A (en) * | 2010-10-15 | 2012-05-09 | 华中农业大学 | Reagent composition for separating total RNA in plant or microorganism and preparation method thereof |
| CN102443580B (en) * | 2010-10-15 | 2013-11-13 | 华中农业大学 | Reagent composition for separating total RNA in plant or microorganism and preparation method thereof |
| WO2021245660A1 (en) * | 2020-05-31 | 2021-12-09 | Ofek Eshkolot Research And Development Ltd. | Compositon for sampling body fluids and secretions for detecting pathogenic agents nucleic acids and for disinfection |
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Open date: 20070131 |