CN117603962A - Universal method for co-extracting DNA and RNA in sample - Google Patents
Universal method for co-extracting DNA and RNA in sample Download PDFInfo
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- JGDITNMASUZKPW-UHFFFAOYSA-K aluminium trichloride hexahydrate Chemical compound O.O.O.O.O.O.Cl[Al](Cl)Cl JGDITNMASUZKPW-UHFFFAOYSA-K 0.000 claims description 3
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Abstract
The invention relates to the technical field of DNA and RNA co-extraction, and discloses a universal method for co-extracting DNA and RNA in a sample, which comprises the following steps: 1) Preparing a column membrane, 2) cracking, and adding a buffer solution 1A and a buffer solution 1B into the cracking medium pipe; 3) Removing impurities, 4) DNA/RNA separation, 5) RNA purification, 6) DNA purification, 7) rinsing 8) elution, and transferring the column to a DNA and RNA recovery tube; the filtrate collected was RNA/DNA for downstream applications; the invention simplifies the operation steps, shortens the operation time, reduces the operation errors and improves the extraction effect; the yield and the purity are effectively improved, the potential pollution risk is reduced, and the accuracy and the reliability of the result are improved; the method is suitable for high-throughput treatment, and meets the requirements of large-scale sample treatment; is applicable to most samples.
Description
Technical Field
The invention relates to the technical field of DNA and RNA co-extraction, in particular to a universal method for co-extracting DNA and RNA in a sample.
Background
With the progress and development of technology, DNA/RNA extraction is required for samples in the fields of biomedicine, genomics, drug development, agricultural science, etc., so co-extraction of DNA and RNA is often required; the DNA and RNA co-extraction technology has important practical value and can provide more comprehensive information and convenience for the fields of basic scientific research, medical diagnosis, drug development and the like.
The development of DNA and RNA co-extraction techniques has been ongoing to meet the needs of researchers for simultaneous DNA and RNA acquisition. The existing society can make remarkable progress in terms of simplifying operation, improving efficiency, guaranteeing quality and the like of a DNA and RNA co-extraction technology. The continued development of these technologies has provided more possibilities for molecular biology research, clinical diagnostics, and personalized medicine, and has facilitated a comprehensive understanding of the genome and transcriptome.
Although DNA and RNA co-extraction techniques are widely used in molecular biology research, there are also some drawbacks and limitations. The following are some of the disadvantages of common DNA and RNA co-extraction techniques:
1. complicated operation: conventional DNA and RNA co-extraction methods typically require multiple steps and complex experimental manipulations, including cell disruption, nucleic acid isolation, washing, and purification, among others. These steps require a lot of time and effort, and are prone to operational errors, affecting the extraction result.
2. Low yield and low purity: traditional methods may result in loss and degradation of DNA/RNA during extraction, resulting in lower yields and poor purity of the extraction. In particular, for some samples that are difficult to lyse (e.g., hard tissue), the effect of conventional methods is more limited.
3. Potential contamination risk: the organic solvents and reagents used in conventional methods may introduce potential contaminants, for example, organic solvents such as phenol/chloroform may contain hazardous substances. These contaminants can interfere with subsequent experiments and analyses, particularly with low abundance target nucleic acids, affecting the accuracy and reliability of the results.
4. Unsuitable for high throughput processing: conventional methods are typically manual and are suitable for small scale sample processing. In large scale sample processing, batch processing may be required, increasing the complexity and time cost of the experiment. With the development of high-throughput technology and the increase of demands, the conventional method cannot meet the requirements of large-scale sample processing, and is low in efficiency and easy to cause operation errors.
5. Sample limitation: some DNA and RNA co-extraction methods have limitations on sample throughput, on the type of sample extracted, and are often focused on samples such as cells and tissues, and on other sample types, such as environmental samples like soil, feces, and body fluid samples such as blood, urine, saliva, and the like, have limited throughput.
From the above description, it is known that there is a strong need for a general method for co-extracting DNA and RNA from a sample to solve the problems.
Disclosure of Invention
The invention provides a general method for co-extracting DNA and RNA in a sample to solve the problems.
The scheme of the invention is as follows:
a universal method for co-extracting DNA and RNA in a sample, comprising the steps of:
1) Preparation of column film
Respectively adding a DNA extraction column and an RNA extraction column into a balance buffer solution, waiting for more than or equal to 1min, centrifuging at maximum speed for 10s, and respectively transferring a silica gel column membrane into a corresponding new collecting pipe;
2) Cleavage of
Weighing the sample and adding the sample into a cracking medium tube; adding the buffer solution 1A and the buffer solution 1B into the cracking medium pipe; cracking by a device; centrifuging by using equipment; the use of buffer 1A and buffer 1B avoids the use of phenol/chloroform cleavage;
3) Impurity removal
Transferring the supernatant into a 2.0 mL micro centrifuge tube; adding buffer No. 2 to precipitate pollutants, swirling for 1s, and centrifuging at maximum speed for 2min;
4) DNA/RNA isolation
Transferring the supernatant to a 2.0 mL microcentrifuge tube, adding buffer solution No. 3, and swirling for 1s to obtain solution A; the volume ratio of the supernatant to the buffer solution No. 3 is 1:1;
adding the solution A into a DNA extraction column, centrifuging, and purifying the filtrate for RNA;
5) RNA purification
Adding buffer solution No. 4 into the filtrate, and swirling for 1s to obtain solution B; the volume ratio of the supernatant to the buffer solution No. 3 to the buffer solution No. 4 is 1:1:1;
adding the solution B into an RNA extraction column, centrifuging for 10s, and discarding filtrate; repeating the steps until all the columns are crossed;
transferring the RNA extraction column into a new RNA collection tube; preparing for rinsing;
6) Purification of DNA:
the DNA extraction column closes the collection tube cover and prepares for rinsing;
7) Rinsing
Adding buffer solution No. 5 in the center of the column, centrifuging, and discarding the filtrate;
adding buffer solution No. 6 in the center of the column, centrifuging, and discarding the filtrate;
adding buffer solution No. 6 in the center of the column, and centrifuging for 30s;
placing the column into a new collecting pipe, and centrifuging at maximum speed for 1min;
8) Elution
Transferring the matched DNA and RNA recovery tube from the column;
adding enzyme-free sterile water into the center of the column, and centrifuging at maximum speed for 1min; the filtrates collected were RNA and DNA, respectively, for downstream applications.
As a preferred embodiment, the step 5) is performed by a RNA extraction column in the obtained RNA collection tube and the step 6) is performed by a DNA extraction column in the obtained DNA collection tube, respectively.
As a preferable technical scheme, in the step 2), the buffer solution 1A and the buffer solution 1B are added into the cracking medium tube, and beta-mercaptoethanol is also added.
As a preferable technical scheme, the cracking medium in the cracking medium pipe is a mixture of zirconia beads and quartz sand.
As a preferable technical scheme, the preparation method of the buffer solution 1A comprises the following steps:
1) Weighing surfactant, sodium dihydrogen phosphate and EDTA-2Na into a liquid preparation barrel, adding diethyl pyrocarbonate treated water into the liquid preparation barrel, stirring for more than or equal to 10 minutes until the solid is dissolved, and clarifying and transparentizing the solution;
2) Testing the pH value of the solution by using a pH meter, wherein the pH value is 3.7-4.3; the pH of the solution was adjusted to 6.0 to 6.6 using sodium hydroxide solution to obtain buffer 1A.
As a preferable technical scheme, the surfactant is one of sodium dodecyl sulfate, triton X-100 and Tween 20.
As a preferable technical scheme, the preparation method of the buffer solution 1B comprises the following steps:
1) Weighing sodium thiocyanate solution and sodium dihydrogen phosphate, and putting the sodium thiocyanate solution and the sodium dihydrogen phosphate into a liquid preparation barrel; adding diethyl pyrocarbonate treated water into a liquid preparation barrel, stirring for more than or equal to 10 minutes until the solid is dissolved, and clarifying and transparentizing the solution;
2) Testing the pH value of the solution by using a pH meter, wherein the pH value is 3.4-4.0; and regulating the pH value of the solution to 7.4-8.0 by using a sodium hydroxide solution, adding a surfactant into the solution after the pH value is regulated, and stirring until the surfactant is dissolved to obtain the buffer solution 1B.
As a preferable technical scheme, the surfactant is one of sodium dodecyl sulfate, triton X-100 and Tween 20.
As a preferable technical scheme, the preparation method of the buffer solution No. 2 comprises the following steps:
1) Weighing aluminum chloride hexahydrate and ammonium acetate into a liquid preparation barrel; adding diethyl pyrocarbonate treated water into a liquid preparation barrel, stirring for more than or equal to 10 minutes until the solid is dissolved, and clarifying and transparentizing the solution;
2) Testing the pH value of the solution by a pH meter, wherein the pH value is 6.5-7.1, obtaining buffer solution No. 2, and preserving at 2-8 ℃ after preparation; the dosage of the buffer solution No. 2 is 300 [ mu ] L/sample.
As a preferable technical scheme, the preparation method of the buffer solution No. 3 comprises the following steps:
1) Weighing guanidine thiocyanate and Tris-HCl, and putting the guanidine thiocyanate and Tris-HCl into a liquid preparation barrel; adding diethyl pyrocarbonate treated water into a liquid preparation barrel, heating and dissolving in a water bath at 45 ℃, stirring for more than or equal to 10 minutes until solid is dissolved, and clarifying and transparentizing the solution;
2) Testing the pH value of the solution by using a pH meter, wherein the pH value is 4.3-4.9; and regulating the pH value of the solution to 6.0-6.6 by using sodium hydroxide solution to obtain buffer solution No. 3.
As a preferable technical scheme, the preparation method of the buffer solution No. 4 comprises the following steps: 1) Weighing guanidine thiocyanate and Tris-HCl, and putting the guanidine thiocyanate and Tris-HCl into a liquid preparation barrel; adding diethyl pyrocarbonate treated water into a liquid preparation barrel, stirring, heating in water bath, and dissolving; stirring for more than or equal to 10 minutes until the solid is dissolved, and the solution is clear and transparent;
2) Testing the pH value of the solution by using a pH meter, wherein the pH value is 4.3-4.9; adjusting the pH value of the solution to 6.0-6.6 by using sodium hydroxide solution;
3) Isopropanol was added to the solution at a ratio of 40-60% to obtain buffer No. 4.
As a preferred technical scheme, the preparation method of the buffer solution No. 5 comprises the following steps:
1) Weighing guanidine thiocyanate and Tris-HCl, and putting the guanidine thiocyanate and Tris-HCl into a liquid preparation barrel; adding diethyl pyrocarbonate treated water into a liquid preparation barrel, stirring, heating in water bath for dissolving, stirring for more than or equal to 10 minutes until solid is dissolved, and clarifying and transparentizing the solution;
2) Testing the pH value of the solution by a pH meter, wherein the pH value is 4.3-4.9; adjusting the pH value of the solution to 7.0-7.6 by using sodium hydroxide solution;
3) Ethanol is added into the solution according to the proportion of 10 to 30 percent to obtain buffer solution No. 5.
As a preferred technical scheme, the preparation method of the buffer solution No. 6 comprises the following steps:
1) Weighing Tris-HCl and Tris, and putting the Tris-HCl and the Tris into a plastic liquid preparation barrel; adding diethyl pyrocarbonate treated water into a liquid preparation barrel, stirring for more than or equal to 10 minutes until the solid is dissolved, and clarifying and transparentizing the solution;
2) Testing the pH value of the solution by a pH meter, wherein the pH value is 6.2-6.8; adjusting the pH value of the solution to 7.0-7.6 by using sodium hydroxide solution;
3) Ethanol is added into the solution according to the proportion of 70-90 percent to obtain buffer solution No. 6.
As a preferable embodiment, the suitable sample includes any one of soil, feces, blood, urine, and saliva.
Due to the adoption of the technical scheme, the universal method for co-extracting DNA and RNA in the sample comprises the following steps of: 1) Preparing column membranes, respectively adding a DNA extraction column and an RNA extraction column into a balance buffer solution, waiting for more than or equal to 1min, centrifuging at maximum speed for 10s, and respectively transferring the silica gel column membranes into corresponding new collecting pipes; 2) Lysing, weighing a sample and adding the sample into a lysis medium tube; adding the buffer solution 1A and the buffer solution 1B into the cracking medium pipe; cracking by a device; centrifuging by using equipment; 3) Removing impurities, taking supernatant and transferring the supernatant into a 2.0 mL micro centrifuge tube; adding buffer No. 2 to precipitate pollutants, swirling for 1s, and centrifuging at maximum speed for 2min; 4) DNA/RNA separation, transferring the supernatant to a 2.0 mL micro centrifuge tube, adding buffer No. 3, and swirling for 1s to obtain solution A; the volume ratio of the supernatant to the buffer solution No. 3 is 1:1; adding the solution A into a DNA extraction column, centrifuging, and purifying the filtrate for RNA; 5) Purifying RNA, adding buffer solution No. 4 into the filtrate, and swirling for 1s to obtain solution B; the volume ratio of the supernatant to the buffer solution No. 3 to the buffer solution No. 4 is 1:1:1; adding the solution B into an RNA extraction column, centrifuging for 10s, and discarding filtrate; repeating the steps until all the columns are crossed; transferring the RNA extraction column into a new RNA collection tube; preparing for rinsing; 6) DNA purification, wherein the DNA extraction column closes the cover of the collecting pipe and is ready for rinsing; 7) Rinsing, adding buffer No. 5 into the center of the column, centrifuging, and discarding the filtrate; adding buffer solution No. 6 in the center of the column, centrifuging, and discarding the filtrate; adding buffer solution No. 6 in the center of the column, and centrifuging for 30s; placing the column into a new collecting pipe, and centrifuging at maximum speed for 1min; 8) Eluting, transferring the column to a matched DNA and RNA recovery tube; adding enzyme-free sterile water into the center of the column, and centrifuging at maximum speed for 1min; the filtrates collected were RNA and DNA, respectively, for downstream applications.
The invention has the advantages that:
the invention simplifies the operation steps, shortens the operation time, reduces the operation errors and improves the extraction effect; the yield and the purity are effectively improved, the potential pollution risk is reduced, and the accuracy and the reliability of the result are improved; the method is suitable for high-throughput treatment, and meets the requirements of large-scale sample treatment; is applicable to most samples.
Drawings
FIG. 1 is a graph showing DNA-Nano concentration data for different amounts of test buffer No. 2 according to example 1 of the present invention;
FIG. 2 is a graph showing the data of test buffer No. 2 of example 1 of the present invention with different amounts of DNA-260/280;
FIG. 3 is a graph showing the data of test buffer No. 2 of example 1 of the present invention with different amounts of DNA-260/230;
FIG. 4 is an electrophoretogram of 250mg SG4 soil DNA extracted with different amounts of test buffer No. 2 according to example 1 of the present invention;
FIG. 5 is an electrophoretogram of 500mg SG4 soil DNA extracted with different amounts of test buffer No. 2 according to example 1 of the present invention;
FIG. 6 is a graph showing RNA-Nano concentration data for different amounts of test buffer No. 2 according to example 1 of the present invention;
FIG. 7 is a graph showing the data of test buffer No. 2 of the present invention for RNA-260/280 at various amounts;
FIG. 8 is a graph showing the data of test buffer No. 2 of the present invention for RNA-260/230 at various amounts;
FIG. 9 is an electrophoretogram of 250mg SG4 soil RNA extracted with different amounts of test buffer No. 2 according to example 1 of the present invention;
FIG. 10 is an electrophoretogram of 500mg SG4 soil RNA extracted from test buffer No. 2 according to example 1 of the present invention at different amounts;
FIG. 11 is a chart showing the data of the extraction of DNA and RNA according to the invention with different amounts of test buffer No. 2 and different centrifugation times after addition of buffer No. 2;
FIG. 12 is an electrophoretogram of the test buffer No. 2 of example 1 of the present invention showing the effect of different amounts of test buffer No. 2 and different centrifugation times after addition of buffer No. 2 on DNA;
FIG. 13 is an electrophoretogram of the test buffer No. 2 of example 1 of the present invention showing the effect of different amounts of test buffer No. 2 and different centrifugation times after addition of buffer No. 2 on RNA;
FIG. 14 is a graph showing the Nano concentration data of DNA and RNA at different amounts of test buffer No. 2 and at different centrifugation times after addition of buffer No. 2 according to example 1 of the present invention;
FIG. 15 is a graph of A260/A280 data of test buffer No. 2 and different centrifugation times after addition of buffer No. 2 versus DNA and RNA according to example 1 of the present invention;
FIG. 16 is a graph of A260/A230 data of test buffer No. 2 and different centrifugation times after addition of buffer No. 2 for DNA and RNA according to example 1 of the present invention;
FIG. 17 is a graph showing data of DNA extraction test in example 1 of the present invention;
FIG. 18 is a graph showing the effect of different proportions of isopropanol in test buffer No. 4 on RNA in example 1 of the present invention;
FIG. 19 is an electrophoretogram of the test pair DNA extraction in example 1 of the present invention;
FIG. 20 is an electrophoretogram of the effect of different ratios of isopropanol in test buffer No. 4 on RNA in example 1 of the present invention;
FIG. 21 is a graph showing the effect of different DNA extraction columns on DNA and RNA extraction in example 1 of the present invention;
FIG. 22 is an electrophoretogram of the invention in example 1 for testing the effect of different DNA extraction columns on DNA extraction;
FIG. 23 is an electrophoretogram of the invention in example 1 for testing the effect of different DNA extraction columns on RNA extraction;
FIG. 24 is a graph showing Nano concentration data for DNA and RNA extraction by different DNA extraction columns according to example 1, wherein the numbers in the groups on the horizontal axis correspond to the corresponding number groups set in FIG. 22, respectively;
FIG. 25 is a graph of A260/A280 data of different DNA extraction columns for DNA and RNA extraction in example 1 of the present invention, wherein the numbers in the groups on the horizontal axis correspond to the corresponding number groups set in FIG. 22, respectively;
FIG. 26 is a graph of A260/A230 data of different DNA extraction columns for DNA and RNA extraction in example 1 of the present invention, wherein the numbers in the groups on the horizontal axis correspond to the corresponding number groups set in FIG. 22, respectively;
FIG. 27 is a graph showing the effect of testing different RNA extraction columns on DNA and RNA extraction in example 1 of the present invention;
FIG. 28 is an electrophoretogram of the effect of testing different RNA extraction columns on RNA extraction in example 1 of the present invention;
FIG. 29 is an electrophoretogram of the test pair DNA extraction in example 1 of the present invention;
FIG. 30 is a graph showing the Nano concentration data of DNA and RNA extraction by different RNA extraction columns according to example 1 of the present invention, wherein the numbers in the groups on the horizontal axis correspond to the corresponding number groups set in FIG. 28, respectively;
FIG. 31 is a graph of A260/A280 data of the different RNA extraction columns tested for DNA and RNA extraction in example 1 of the present invention, wherein the numbers in the groups on the horizontal axis correspond to the corresponding number groups set forth in FIG. 22, respectively;
FIG. 32 is a graph of A260/A230 data of the different RNA extraction columns tested for DNA and RNA extraction in example 1 of the present invention, wherein the numbers in the groups on the horizontal axis correspond to the corresponding number groups set forth in FIG. 22, respectively;
FIG. 33 is a graph showing the difference between the Nano concentration data obtained by extracting DNA and RNA from the same sample in accordance with the test example 1 of the present invention and the comparison example;
FIG. 34 is a graph showing the difference between the data obtained in the A260/A280 of the invention test example 1 and the data obtained in the comparison example for the extraction of DNA and RNA from the same sample;
FIG. 35 is a graph showing the difference between the data of A260/A230 of the invention test example 1 and the data of the comparative example for the extraction of DNA and RNA from the same sample;
FIG. 36 is a diagram showing the difference in electrophoresis of DNA extraction of the same sample in test example 1 and comparative example according to the present invention;
FIG. 37 is a graph showing the difference in electrophoresis of RNA extraction from the same sample in test example 1 and comparative example according to the present invention;
FIG. 38 is a graph showing the difference between Nano concentration data obtained by DNA extraction in test example 1 and comparative example according to the present invention;
FIG. 39 is a graph showing the difference between Nano concentration data obtained by extracting RNA in test example 1 and comparative example according to the present invention;
FIG. 40 is a diagram showing the difference in electrophoresis between DNA and RNA extraction according to the test example 1 and the comparative example of the present invention;
FIG. 41 is a graph showing the difference between the data of A260/A280 obtained in the test example 1 and the data obtained in the comparative example;
FIG. 42 is a graph showing the difference between the data of A260/A230 of the test example 1 and the comparative example;
FIG. 43 is a graph showing the difference between the data of A260/A280 obtained by the test example 1 and the data obtained by the comparison of RNA extraction according to the present invention;
FIG. 44 is a graph showing the difference between the data of A260/A230 of the test example 1 and the comparative example for RNA extraction according to the present invention;
FIG. 45 is a graph showing the difference between Nano concentration data obtained by extracting DNA and RNA according to test example 2 and comparative example of the present invention;
FIG. 46 is a graph showing the difference between data obtained in A260/A280 of the test example 2 and the comparative example for DNA and RNA extraction according to the present invention;
FIG. 47 is a graph showing the difference between data obtained in A260/A230 of the test example 2 and the comparative example for DNA and RNA extraction according to the present invention;
FIG. 48 is a diagram showing the difference between the electrophoresis of DNA and RNA extraction according to the test example 2 and the comparative example of the present invention.
Detailed Description
The invention provides a universal method for co-extracting DNA and RNA in a sample.
The invention is further described in connection with the following embodiments in order to make the technical means, the creation features, the achievement of the purpose and the effect of the invention easy to understand.
1) Preparation of column film
Respectively adding a DNA extraction column and an RNA extraction column into a balance buffer solution, waiting for more than or equal to 1min, centrifuging at maximum speed for 10s, and respectively transferring a silica gel column membrane into a corresponding new collecting pipe;
2) Cleavage of
Weighing the sample and adding the sample into a cracking medium tube; 150. Mu.L of buffer 1A and 600. Mu.L of buffer 1B were added to the lysis medium tube (8. Mu.L of beta. -mercaptoethanol may be added if nucleic acid integrity is a concern); vortex at 2500-2700 rpm for 15 min or crack the equipment at 5m/sec for 35sec; centrifuging at maximum speed for 2min;
3) Impurity removal
Transferring the supernatant into a 2.0 mL micro centrifuge tube; adding 300 mu L of buffer No. 2 to precipitate pollutants, swirling for 1s, and centrifuging at a maximum speed for 2min;
4) DNA/RNA isolation
Transferring the supernatant to a 2.0 mL micro centrifuge tube, adding 1 volume of buffer solution No. 3, and swirling for 1s to obtain solution A; the volume ratio of the supernatant to the buffer solution No. 3 is 1:1;
adding 700 mu L of the solution A into the DNA extraction column, centrifuging at 15000rpm for 10s, collecting filtrate, and covering a collecting pipe cover filled with the filtrate;
placing the DNA extraction column into a new DNA collecting pipe, adding the rest solution A into the DNA extraction column, centrifuging at 15000rpm for 10s, collecting filtrate, and covering a collecting pipe cover with the filtrate;
5) RNA purification
Adding buffer solution No. 4 with the volume of the supernatant 1 in the step 4) into the filtrate, and swirling for 1s to obtain solution B; the volume ratio of the supernatant to the buffer solution No. 3 to the buffer solution No. 4 is 1:1:1;
adding 800 mu L of the solution B into an RNA extraction column, centrifuging at 15000rpm for 10s, and discarding the filtrate; repeating the steps until all the columns are crossed;
transferring the RNA extraction column into a new RNA collection tube, and preparing for rinsing;
6) Purification of DNA:
the DNA extraction column closes the collection tube cover and prepares for rinsing;
7) Rinsing
Adding 700 mu L of buffer No. 5 into the centers of the DNA extraction column and the RNA extraction column respectively, centrifuging at 15000rpm for 10s, and discarding the filtrate;
adding 700 mu L of buffer solution No. 6 to the centers of the DNA extraction column and the RNA extraction column respectively, centrifuging at 15000rpm for 10s, and discarding the filtrate;
adding 500 μl buffer No. 6 to the centers of the DNA extraction column and RNA extraction column, and centrifuging at 15000rpm for 30s;
respectively placing the DNA extraction column and the RNA extraction column into a new collecting pipe, and centrifuging at maximum speed for 1min;
8) Elution
Transferring the DNA extraction column and the RNA extraction column to a DNA collecting pipe and an RNA collecting pipe respectively;
respectively adding 100 mu L of sterile water without enzyme into the centers of the DNA extraction column and the RNA extraction column, and centrifuging at maximum speed for 1min; the filtrates collected were DNA and RNA, respectively, for downstream applications.
The cracking medium in the cracking medium pipe is a mixture of zirconia beads and quartz sand.
Cleavage Medium formulation Table
The sample is 250-500 mg of soil (SG 4).
1. Preparation of diethyl pyrocarbonate treated water
Table 1:
(1) Weighing purified water, adding the purified water into a glass liquid preparation bottle, accurately extracting DEPC liquid by a liquid transferring gun, adding the DEPC liquid into the weighed purified water, covering a bottle cap, horizontally placing the glass liquid preparation bottle on a table top, rapidly shaking until a large amount of white bubbles are generated, continuously shaking for 2 minutes, rapidly eliminating the bubbles after the glass liquid preparation bottle is erected, visually completely fusing DEPC into water, and standing for more than 12 hours without obvious DEPC wall marks on the bottle body.
(2) After the solution stands still, high-temperature sterilization treatment is needed. After the liquid preparation bottle cap is unscrewed, the liquid preparation bottle is put into a high-temperature sterilization pot for sterilization at 121 ℃ for 30 minutes. After the preparation is finished, the cover is covered for storage and used within two weeks, and the cover is expired for being used.
2. Buffer 1A formulation
Table 2:
(1) Weighing surfactant, sodium dihydrogen phosphate and EDTA-2Na into a liquid preparation barrel, adding diethyl pyrocarbonate treated water into the liquid preparation barrel, and stirring for at least 10 min until the solid is dissolved, wherein the solution is clear and transparent. The surfactant is sodium dodecyl sulfate.
(2) The pH of the solution was measured with a pH meter and should be 3.7-4.3 at 25 ℃. The pH of the solution was adjusted to 6.0-6.6 using sodium hydroxide solution.
3. Buffer 1B preparation
Table 3:
(1) Weighing sodium thiocyanate solution and sodium dihydrogen phosphate, and placing into a liquid preparation barrel. Adding diethyl pyrocarbonate treated water into a liquid preparation barrel, and stirring for at least 10 minutes until the solid is dissolved, wherein the solution is clear and transparent.
(2) The pH value of the solution is measured by a pH meter, and the pH value (25 ℃) is 3.4-4.0. NaOH solution is used for adjusting the pH value of the solution to 7.4-8.0. After the pH is adjusted, a surfactant is added into the solution and stirred until the surfactant is dissolved. The surfactant is Triton X-100.
4. Buffer solution No. 2 preparation
Table 4:
(1) The aluminum chloride hexahydrate and the ammonium acetate are weighed into a liquid preparation barrel. Adding diethyl pyrocarbonate treated water into a liquid preparation barrel, and stirring for at least 10 minutes until the solid is dissolved, wherein the solution is clear and transparent.
(2) The pH of the solution was measured with a pH meter and should be 6.5-7.1 (25 ℃ C.) without adjusting the pH of the solution. Note that: after being prepared, the mixture needs to be stored at the temperature of 2-8 ℃.
Four different amounts of 150/200/250/300 [ mu ] L of buffer No. 2 were tested during the 300 [ mu ] L/sample procedure, respectively, and the yields of extracted DNA and RNA decreased to different extents with increasing amounts of use, but the purity increased, and it was verified that the centrifugation time in this step was increased from 1 minute to 2 minutes, and the yields increased and the high purity was maintained with 300 [ mu ] L/sample (see FIGS. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16 for details)
5. Buffer 3 preparation
Table 5:
(1) Weighing guanidine thiocyanate and Tris-HCl, and placing the guanidine thiocyanate and Tris-HCl into a liquid preparation barrel. Adding diethyl pyrocarbonate treated water into a liquid preparation barrel, and stirring for at least 10 minutes until the solid is dissolved, wherein the solution is clear and transparent.
(2) The pH value of the solution is measured by a pH meter, and the pH value (25 ℃) is 4.3-4.9. NaOH solution is used for adjusting the pH value of the solution to 6.0-6.6.
6. Buffer No. 4 preparation
Table 6:
(1) Weighing guanidine thiocyanate and Tris-HCl, and placing the guanidine thiocyanate and Tris-HCl into a liquid preparation barrel. Adding diethyl pyrocarbonate treated water into a liquid preparation barrel, and stirring for at least 10 minutes until the solid is dissolved, wherein the solution is clear and transparent.
(2) The pH value of the solution is measured by a pH meter, and the pH value (25 ℃) is 4.3-4.9. NaOH solution is used for adjusting the pH value of the solution to 6.0-6.6.
(3) Isopropanol was added to the solution at a ratio of 40-60%. The effect is shown in detail in fig. 17, 18, 19 and 20.
7. Buffer 5 preparation
Table 7:
(1) Weighing guanidine thiocyanate and Tris-HCl, and placing the guanidine thiocyanate and Tris-HCl into a liquid preparation barrel. Adding diethyl pyrocarbonate treated water into a liquid preparation barrel, and stirring for at least 10 minutes until the solid is dissolved, wherein the solution is clear and transparent.
(2) The pH value of the solution is measured by a pH meter, and the pH value (25 ℃) is 4.3-4.9. NaOH solution is used for adjusting the pH value of the solution to 7.0-7.6.
(3) Ethanol is added into the solution according to the proportion of 10 to 30 percent.
8. Buffer 6 preparation
Table 8:
(1) And weighing Tris-HCl and Tris by an electronic scale, and putting the Tris-HCl and Tris into a plastic liquid preparation barrel. Adding diethyl pyrocarbonate treated water into a liquid preparation barrel, starting an electric stirrer, and stirring for at least 10 minutes until the solid is dissolved, wherein the solution is clear and transparent.
(2) The pH of the solution was measured with a pH meter and should be 6.2 to 6.8 at a pH (25 ℃). NaOH solution is used for adjusting the pH value of the solution to 7.0-7.6.
(3) Ethanol is added into the solution according to the proportion of 70-90 percent.
9. Asepsis water without enzyme
Liquid preparation table 1:
10. balanced buffer preparation
Table 9:
(1) Weighing sodium hydroxide, placing into a liquid preparation barrel, adding diethyl pyrocarbonate treated water, stirring for at least 10 minutes until the solid is dissolved, and clarifying and transparentizing the solution.
The DNA extraction Column is DNA Column, and the RNA extraction Column is RNA Column. And the silica gel column membrane can efficiently separate DNA and RNA.
Comparative experiment 1
Example 1 preparation measurement yield and purity are example groups;
comparative example 1, following the procedure of example 1, the only difference was that the DNA extraction column was green, and the yield and purity were measured;
comparative example 2, according to the procedure of example 1, with the only difference that the DNA extraction column was Jie Tee, yield and purity were measured;
comparative example 3 the yield and purity were measured according to the procedure of example 1, with the only difference that the DNA extraction column was Magen;
in contrast, the results revealed that the yields of the workers and the Jie Tee were comparable to those of the currently used DNA Columb, and that the Magen yields were low, but the yields were finally determined to be the currently used DNA Columb in view of the purity of the DNA, in view of the cost and stability. (see FIGS. 21, 22, 23, 24, 25 and 26).
The RNA extraction columns were compared and the RNA Column currently used was finally selected. The selected RNA Columbn was identical to the new one in terms of yield and purity results, and the stability of the selected RNA Columbn was better (see FIGS. 27, 28, 29, 30, 31 and 32).
Comparative experiment 4
Using each buffer prepared in the present application, the DNA and RNA extraction columns were used in this example 1 (group of examples shown in the drawing) and comparative example 4 (Zymo shown in the drawing), respectively, and from the results, it was found that the extraction of DNA by the comparative example DNA extraction column membrane was incomplete, resulting in significant DNA residues in the extracted RNA, which did not meet the purpose of DNA and RNA separation (see FIG. 33, FIG. 34, FIG. 35, FIG. 36 and FIG. 37).
Comparative experiment 5
Soil samples of 250mg were extracted in example 1, and comparative example 5 was extracted using Zymo product, and after both extraction, the yield and purity were measured, respectively, and the results are shown in fig. 38, 39, 40, 41, 42, 43 and 44.
Example 2
The sample is an extracted blood sample 200 [ mu ] L.
1) Preparation of column film
Respectively adding a DNA extraction column and an RNA extraction column into a balance buffer solution, waiting for more than or equal to 1min, centrifuging at maximum speed for 10s, and respectively transferring a silica gel column membrane into a corresponding new collecting pipe;
2) Cleavage of
Weighing the sample and adding the sample into a cracking medium tube; 150. Mu.L of buffer 1A and 600. Mu.L of buffer 1B were added to the lysis medium tube; (if there is concern about nucleic acid integrity, 8. Mu.L of beta-mercaptoethanol may be added); vortex at 2500-2700 rpm for 15 min or crack the equipment at 5m/sec for 35sec; centrifuging at maximum speed for 2min;
3) Impurity removal
Transferring the supernatant into a 2.0 mL micro centrifuge tube; adding 300 mu L of buffer No. 2 to precipitate pollutants, swirling for 1s, and centrifuging at a maximum speed for 2min;
4) DNA/RNA isolation
Transferring the supernatant to a 2.0 mL micro centrifuge tube, adding 1 volume of buffer solution No. 3, and swirling for 1s to obtain solution A; the volume ratio of the supernatant to the buffer solution No. 3 is 1:1;
adding 700 mu L of the solution A into the DNA extraction column, centrifuging at 15000rpm for 10s, collecting filtrate, and covering a collecting pipe cover filled with the filtrate;
placing the DNA extraction column into a new DNA collecting pipe, adding the rest solution A into the DNA extraction column, centrifuging at 15000rpm for 10s, collecting filtrate, and covering a collecting pipe cover with the filtrate;
5) RNA purification
Adding buffer solution No. 4 with the volume of the supernatant 1 in the step 4) into the filtrate, and swirling for 1s to obtain solution B; the volume ratio of the supernatant to the buffer solution No. 3 to the buffer solution No. 4 is 1:1:1;
adding 800 mu L of the solution B into an RNA extraction column, centrifuging at 15000rpm for 10s, and discarding the filtrate; repeating the steps until all the columns are crossed;
transferring the RNA extraction column into a new RNA collection tube, and preparing for rinsing;
6) Purification of DNA:
the DNA extraction column closes the collection tube cover and prepares for rinsing;
7) Rinsing
Adding 700 mu L of buffer No. 5 into the centers of the DNA extraction column and the RNA extraction column respectively, centrifuging at 15000rpm for 10s, and discarding the filtrate;
adding 700 mu L of buffer solution No. 6 to the centers of the DNA extraction column and the RNA extraction column respectively, centrifuging at 15000rpm for 10s, and discarding the filtrate;
adding 500 μl buffer No. 6 to the centers of the DNA extraction column and RNA extraction column, and centrifuging at 15000rpm for 30s;
respectively placing the DNA extraction column and the RNA extraction column into a new collecting pipe, and centrifuging at maximum speed for 1min;
8) Elution
Transferring the DNA extraction column and the RNA extraction column to a DNA collecting pipe and an RNA collecting pipe respectively;
respectively adding 100 mu L of sterile water without enzyme into the centers of the DNA extraction column and the RNA extraction column, and centrifuging at maximum speed for 1min; the filtrates collected were DNA and RNA, respectively, for downstream applications.
The cracking medium in the cracking medium pipe is a mixture of zirconia beads and quartz sand.
The preparation of lysis medium, diethyl pyrocarbonate treatment water, buffer 1A, buffer 1B, buffer 2, buffer 3, buffer 4, buffer 5, buffer 6, sterile water and equilibration buffer were the same as each of the formulations in example 1.
The DNA extraction Column is DNA Column, and the RNA extraction Column is RNA Column. And the silica gel column membrane can efficiently separate DNA and RNA.
Comparative experiment 6
Blood sample 200 μl was extracted using example 2, comparative example 6 was extracted using Zymo product, and after extraction, the yield and purity were measured, respectively, and the results are shown in fig. 45, 46, 47 and 48.
The foregoing has shown and described the basic principles, main features and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
1. A universal method for co-extracting DNA and RNA in a sample, comprising the steps of:
1) Preparation of column film
Respectively adding a DNA extraction column and an RNA extraction column into a balance buffer solution, waiting for more than or equal to 1min, centrifuging at maximum speed for 10s, and respectively transferring a silica gel column membrane into a corresponding new collecting pipe;
2) Cleavage of
Weighing the sample and adding the sample into a cracking medium tube; adding the buffer solution 1A and the buffer solution 1B into the cracking medium pipe; cracking by a device; centrifuging by using equipment;
3) Impurity removal
Transferring the supernatant into a 2.0 mL micro centrifuge tube; adding buffer No. 2 to precipitate pollutants, swirling for 1s, and centrifuging at maximum speed for 2min;
4) DNA/RNA isolation
Transferring the supernatant to a 2.0 mL microcentrifuge tube, adding buffer solution No. 3, and swirling for 1s to obtain solution A; the volume ratio of the supernatant to the buffer solution No. 3 is 1:1;
adding the solution A into a DNA extraction column, centrifuging, and purifying the filtrate for RNA;
5) RNA purification
Adding buffer solution No. 4 into the filtrate, and swirling for 1s to obtain solution B; the volume ratio of the supernatant to the buffer solution No. 3 to the buffer solution No. 4 is 1:1:1;
adding the solution B into an RNA extraction column, centrifuging for 10s, and discarding filtrate; repeating the steps until all the columns are crossed;
transferring the RNA extraction column into a new RNA collection tube, and preparing for rinsing;
6) DNA purification
The DNA extraction column closes the collection tube cover and prepares for rinsing;
7) Rinsing
Adding buffer solution No. 5 in the center of the column, centrifuging, and discarding the filtrate;
adding buffer solution No. 6 in the center of the column, centrifuging, and discarding the filtrate;
adding buffer solution No. 6 in the center of the column, and centrifuging for 30s;
placing the column into a new collecting pipe, and centrifuging at maximum speed for 1min;
8) Elution
Transferring the column to a corresponding DNA and RNA recovery tube;
adding enzyme-free sterile water into the center of the column, and centrifuging at maximum speed for 1min; the filtrates collected were RNA and DNA, respectively, for downstream applications.
2. The universal method for co-extracting DNA and RNA in a sample according to claim 1, wherein the preparation method of the buffer solution 1A is as follows:
1) Weighing surfactant, sodium dihydrogen phosphate and EDTA-2Na into a liquid preparation barrel, adding diethyl pyrocarbonate treated water into the liquid preparation barrel, stirring for more than or equal to 10 minutes until the solid is dissolved, and clarifying and transparentizing the solution;
2) Testing the pH value of the solution by using a pH meter, wherein the pH value is 3.7-4.3; the pH of the solution was adjusted to 6.0 to 6.6 using sodium hydroxide solution to obtain buffer 1A.
3. The universal method for co-extracting DNA and RNA in a sample according to claim 1, wherein the preparation method of the buffer solution 1B is as follows:
1) Weighing sodium thiocyanate solution and sodium dihydrogen phosphate, and putting the sodium thiocyanate solution and the sodium dihydrogen phosphate into a liquid preparation barrel; adding diethyl pyrocarbonate treated water into a liquid preparation barrel, stirring for more than or equal to 10 minutes until the solid is dissolved, and clarifying and transparentizing the solution;
2) Testing the pH value of the solution by using a pH meter, wherein the pH value is 3.4-4.0; and regulating the pH value of the solution to 7.4-8.0 by using a sodium hydroxide solution, adding a surfactant into the solution after the pH value is regulated, and stirring until the surfactant is dissolved to obtain the buffer solution 1B.
4. The universal method for co-extracting DNA and RNA from a sample according to claim 1, wherein the buffer No. 2 is prepared by:
1) Weighing aluminum chloride hexahydrate and ammonium acetate into a liquid preparation barrel; adding diethyl pyrocarbonate treated water into a liquid preparation barrel, stirring for more than or equal to 10 minutes until the solid is dissolved, and clarifying and transparentizing the solution;
2) Testing the pH value of the solution by a pH meter, wherein the pH value is 6.5-7.1, obtaining buffer solution No. 2, and preserving at 2-8 ℃ after preparation; the dosage of the buffer solution No. 2 is 300 [ mu ] L/sample.
5. The universal method for co-extracting DNA and RNA from a sample according to claim 1, wherein said buffer No. 3 is prepared by:
1) Weighing guanidine thiocyanate and Tris-HCl, and putting the guanidine thiocyanate and Tris-HCl into a liquid preparation barrel; adding diethyl pyrocarbonate treated water into a liquid preparation barrel, heating and dissolving in a water bath at 45 ℃, stirring for more than or equal to 10 minutes until solid is dissolved, and clarifying and transparentizing the solution;
2) Testing the pH value of the solution by using a pH meter, wherein the pH value is 4.3-4.9; and regulating the pH value of the solution to 6.0-6.6 by using sodium hydroxide solution to obtain buffer solution No. 3.
6. The universal method for co-extracting DNA and RNA from a sample according to claim 1, wherein said buffer No. 4 is prepared by:
1) Weighing guanidine thiocyanate and Tris-HCl, and putting the guanidine thiocyanate and Tris-HCl into a liquid preparation barrel; adding diethyl pyrocarbonate treated water into a liquid preparation barrel, stirring, heating in water bath, and dissolving; stirring for more than or equal to 10 minutes until the solid is dissolved, and the solution is clear and transparent;
2) Testing the pH value of the solution by using a pH meter, wherein the pH value is 4.3-4.9; adjusting the pH value of the solution to 6.0-6.6 by using sodium hydroxide solution;
3) Isopropanol was added to the solution at a ratio of 40-60% to obtain buffer No. 4.
7. The universal method for co-extracting DNA and RNA from a sample according to claim 1, wherein said buffer No. 5 is prepared by:
1) Weighing guanidine thiocyanate and Tris-HCl, and putting the guanidine thiocyanate and Tris-HCl into a liquid preparation barrel; adding diethyl pyrocarbonate treated water into a liquid preparation barrel, stirring, heating in water bath for dissolving, stirring for more than or equal to 10 minutes until solid is dissolved, and clarifying and transparentizing the solution;
2) Testing the pH value of the solution by a pH meter, wherein the pH value is 4.3-4.9; adjusting the pH value of the solution to 7.0-7.6 by using sodium hydroxide solution;
3) Ethanol is added into the solution according to the proportion of 10 to 30 percent to obtain buffer solution No. 5.
8. The universal method for co-extracting DNA and RNA from a sample according to claim 1, wherein said buffer No. 6 is prepared by:
1) Weighing Tris-HCl and Tris, and putting the Tris-HCl and the Tris into a plastic liquid preparation barrel; adding diethyl pyrocarbonate treated water into a liquid preparation barrel, stirring for more than or equal to 10 minutes until the solid is dissolved, and clarifying and transparentizing the solution;
2) Testing the pH value of the solution by a pH meter, wherein the pH value is 6.2-6.8; adjusting the pH value of the solution to 7.0-7.6 by using sodium hydroxide solution;
3) Ethanol is added into the solution according to the proportion of 70-90 percent to obtain buffer solution No. 6.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845436A (en) * | 2010-06-28 | 2010-09-29 | 湖南大学 | Method for simultaneously extracting total DNA and RNA from compost |
| CN102812118A (en) * | 2010-04-08 | 2012-12-05 | 恰根有限公司 | Method for isolating and purifying nucleic acids |
| WO2019180519A1 (en) * | 2018-03-17 | 2019-09-26 | Metagenom Bio Holdings Pte Ltd. | Method and kit for isolation of nucleic acids from a sample |
| CN112195177A (en) * | 2020-10-28 | 2021-01-08 | 上海慕柏生物医学科技有限公司 | Nucleic acid extraction method and kit |
| CN112313344A (en) * | 2018-04-24 | 2021-02-02 | 凯杰科技有限公司 | Isolation of nucleic acids and removal of inhibitors from complex samples |
-
2024
- 2024-01-23 CN CN202410089808.5A patent/CN117603962A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102812118A (en) * | 2010-04-08 | 2012-12-05 | 恰根有限公司 | Method for isolating and purifying nucleic acids |
| CN101845436A (en) * | 2010-06-28 | 2010-09-29 | 湖南大学 | Method for simultaneously extracting total DNA and RNA from compost |
| WO2019180519A1 (en) * | 2018-03-17 | 2019-09-26 | Metagenom Bio Holdings Pte Ltd. | Method and kit for isolation of nucleic acids from a sample |
| CN112313344A (en) * | 2018-04-24 | 2021-02-02 | 凯杰科技有限公司 | Isolation of nucleic acids and removal of inhibitors from complex samples |
| CN112195177A (en) * | 2020-10-28 | 2021-01-08 | 上海慕柏生物医学科技有限公司 | Nucleic acid extraction method and kit |
Non-Patent Citations (2)
| Title |
|---|
| E. TOURNIER ET AL.: "Modification of a commercial DNA extraction kit for safe and rapid recovery of DNA and RNA simultaneously from soil, without the use of harmful solvents", 《METHODSX》, vol. 2, 28 March 2015 (2015-03-28), pages 183 * |
| MP BIOMEDICALS: "SPINeasy® DNA/RNA Kit for Soil", 《产品简介》, 31 May 2023 (2023-05-31), pages 7 - 9 * |
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