CN118165982A - Kit for rapidly extracting RNA and application thereof - Google Patents
Kit for rapidly extracting RNA and application thereof Download PDFInfo
- Publication number
- CN118165982A CN118165982A CN202410462002.6A CN202410462002A CN118165982A CN 118165982 A CN118165982 A CN 118165982A CN 202410462002 A CN202410462002 A CN 202410462002A CN 118165982 A CN118165982 A CN 118165982A
- Authority
- CN
- China
- Prior art keywords
- rna
- kit
- lysate
- hcl
- tris
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001179 sorption measurement Methods 0.000 claims abstract description 34
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 30
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000006166 lysate Substances 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 22
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 108010067770 Endopeptidase K Proteins 0.000 claims abstract description 15
- 239000011780 sodium chloride Substances 0.000 claims abstract description 15
- 239000003480 eluent Substances 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 11
- -1 sucrose fatty acid ester Chemical class 0.000 claims abstract description 11
- 229930006000 Sucrose Natural products 0.000 claims abstract description 10
- 239000013504 Triton X-100 Substances 0.000 claims abstract description 10
- 229920004890 Triton X-100 Polymers 0.000 claims abstract description 10
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 10
- 239000000194 fatty acid Substances 0.000 claims abstract description 10
- 229930195729 fatty acid Natural products 0.000 claims abstract description 10
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000005720 sucrose Substances 0.000 claims abstract description 10
- 239000000203 mixture Substances 0.000 claims abstract description 6
- 108010077895 Sarcosine Proteins 0.000 claims abstract description 5
- 229940048098 sodium sarcosinate Drugs 0.000 claims abstract description 5
- 238000000605 extraction Methods 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 102000016911 Deoxyribonucleases Human genes 0.000 claims description 10
- 108010053770 Deoxyribonucleases Proteins 0.000 claims description 10
- 108091092584 GDNA Proteins 0.000 claims description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- 239000011535 reaction buffer Substances 0.000 claims description 8
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- 229910052710 silicon Inorganic materials 0.000 claims description 7
- 239000010703 silicon Substances 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 5
- 230000002934 lysing effect Effects 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- 239000001110 calcium chloride Substances 0.000 claims description 4
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 4
- 239000012498 ultrapure water Substances 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 238000005336 cracking Methods 0.000 claims description 2
- 239000012487 rinsing solution Substances 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 239000012224 working solution Substances 0.000 claims description 2
- IQYRLRYAPJYANI-UHFFFAOYSA-N 2-[dodecyl(methyl)amino]acetic acid;sodium Chemical compound [Na].CCCCCCCCCCCCN(C)CC(O)=O IQYRLRYAPJYANI-UHFFFAOYSA-N 0.000 claims 2
- 238000002123 RNA extraction Methods 0.000 abstract description 27
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 5
- 102000006382 Ribonucleases Human genes 0.000 abstract description 4
- 108010083644 Ribonucleases Proteins 0.000 abstract description 4
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- 238000009472 formulation Methods 0.000 abstract 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 56
- 239000000523 sample Substances 0.000 description 19
- 210000004027 cell Anatomy 0.000 description 15
- 230000000052 comparative effect Effects 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 238000010802 RNA extraction kit Methods 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 6
- 230000001464 adherent effect Effects 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 206010009944 Colon cancer Diseases 0.000 description 4
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 239000008346 aqueous phase Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 210000005228 liver tissue Anatomy 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 241000219194 Arabidopsis Species 0.000 description 3
- 238000000227 grinding Methods 0.000 description 3
- 231100000252 nontoxic Toxicity 0.000 description 3
- 230000003000 nontoxic effect Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 238000012233 TRIzol extraction Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000007886 magnetic bead extraction Methods 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- BMYCCWYAFNPAQC-UHFFFAOYSA-N 2-[dodecyl(methyl)azaniumyl]acetate Chemical compound CCCCCCCCCCCCN(C)CC(O)=O BMYCCWYAFNPAQC-UHFFFAOYSA-N 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108700001907 N-dodecylsarcosinate Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 231100000481 chemical toxicant Toxicity 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 150000002357 guanidines Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003098 myoblast Anatomy 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for rapidly extracting RNA, which comprises a lysate, proteinase K, a rinsing liquid, an eluent, an RNA adsorption column and a collecting pipe, wherein the lysate comprises guanidine isothiocyanate, tris-HCl, N-dodecyl sodium sarcosinate, sodium chloride, sucrose fatty acid ester, triton X-100 and isopropanol. The invention also discloses a method for rapidly extracting RNA by using the kit. The invention can rapidly lyse cells to release RNA and inhibit RNase activity within 1min and rapidly and completely extract RNA by improving the components of the kit, particularly the formulation of the lysate. In addition, the lysate in the kit does not contain toxic reagents such as phenol, chloroform and the like, so that the RNA extraction process can be ensured to be green and safe.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to extraction of RNA and removal of gDNA.
Background
RNA extraction technology is an important technology in the field of molecular biology, and high-purity and high-quality RNA is the basis of experiments such as transcriptome sequencing, cDNA library establishment, RT-PCR, q-PCR, northern blot, microRNA detection, chip analysis, gene cloning and the like. Currently, the main methods for extracting RNA include phenol/chloroform extraction, trizol extraction, silica-based membrane or silica resin extraction, column purification, magnetic bead extraction, etc. The existing RNA extraction kits on the market are generally developed based on the method. The phenol/chloroform extraction method and the Trizol extraction method need toxic chemical reagents such as chloroform, phenol, beta-mercaptoethanol and the like when in use, have hidden danger to operators and environment, and the extraction process takes about 1 hour, which is not beneficial to improving the experimental efficiency. The quality of RNA extracted by a silicon-based membrane or silicon resin extraction method, a column purification method and a magnetic bead extraction method is unstable, the method is not suitable for extracting a small amount of sample RNA, and the operation process is tedious and time-consuming. In summary, how to develop a green, nontoxic, rapid and efficient RNA extraction kit is a major topic in the current RNA extraction technology field.
Disclosure of Invention
One of the technical problems to be solved by the invention is to provide a kit for rapidly extracting RNA, which can rapidly and completely extract RNA, and is safe and nontoxic.
In order to solve the technical problems, the kit for rapidly extracting RNA comprises a lysate, proteinase K, a rinsing liquid, an eluent, an RNA adsorption column and a collecting pipe, wherein the lysate comprises guanidine isothiocyanate, tris-HCl, N-dodecyl sodium sarcosinate, sodium chloride, sucrose fatty acid ester, triton X-100 and isopropanol.
In order to remove gDNA remained in RNA, the kit for rapid extraction of RNA of the invention can further comprise DNase and reaction buffer solution. The dnase is preferably a double-strand specific dnase. The reaction buffer is preferably
10 Xreaction Buffer, comprising Tris-HCl 0.02-0.08M, magnesium sulfate 0.01-0.02M, and calcium chloride 1.0-3.0 mM.
Preferably, the lysate in the RNA rapid extraction kit of the invention preferably contains 4.0-8.0M guanidine isothiocyanate, 0.02-0.15M Tris-HCl with pH of 5.0-6.5, 0.2-2.0% N-dodecyl sodium sarcosinate, 0.2-1.0M sodium chloride, 0.1-0.5% sucrose fatty acid ester, 0.1-1.0% Triton X-100 and 30-50% isopropyl alcohol.
Preferably, the concentration of proteinase K in the kit for rapid RNA extraction of the present invention is preferably 10mg/mL to the maximum
30Mg/mL, >30U/mg; the rinsing liquid preferably contains Tris-HCl 0.02-0.2M with pH of 6.0-8.0, sodium chloride 0.02-0.2M, and absolute ethanol with volume fraction of 60% -80%; the eluent is preferably sterile ultrapure water from which RNase has been removed; the RNA adsorption column is preferably a silicon membrane adsorption column.
The second technical problem to be solved by the invention is to provide the application of the kit in rapid extraction of RNA.
The third technical problem to be solved by the invention is to provide a method for rapidly extracting RNA by using the kit, which comprises the following steps:
1) Lysing the samples: adding lysate and proteinase K into the treated cells or tissues, mixing, and standing at room temperature for reaction;
2) Rinsing impurities: adding a rinsing liquid into the RNA adsorption column, performing air-separation after rinsing, uncovering and standing, and thoroughly removing the rinsing liquid;
3) Eluting RNA: suspending and dripping eluent on the RNA adsorption column, standing at room temperature, centrifuging and collecting filtrate to obtain RNA.
In order to remove the gDNA remaining in the RNA, the following gDNA removal step may be further included between the above step 1) and step 2): transferring the cracking mixture obtained after the reaction into an RNA adsorption column, centrifuging, suspending and dripping a DNase working solution prepared from DNase and a reaction buffer solution on the RNA adsorption column, and reacting at room temperature.
The step of removing gDNA is selected according to the requirement, and the step can be added to completely remove the residual gDNA in RNA without influencing subsequent experiments such as reverse transcription.
The invention uses three surfactants of N-dodecyl sarcosinate, sucrose fatty acid ester and Triton X-100 to lyse cells with proper concentration ratio and release RNA rapidly by improving the component formula of the RNA extraction kit, especially the lysate formula; the high-concentration guanidine isothiocyanate is used for effectively inhibiting the activity of the RNase and protecting RNA from degradation; the Tris-HCl buffer extraction system is used for buffering the pH environment, so that RNA, DNA and protein are ensured to be separated, and the RNA is specifically settled by isopropanol, thereby achieving the purposes of extracting and separating RNA. Thus, under the double action of guanidine salt and surfactant, the efficient cell lysis (only 1min is needed in the whole lysis process) and the inhibition of RNase activity are realized. Compared with the existing RNA extraction kit, the kit has the following advantages and beneficial effects:
1. the method is simple to operate, the whole process is operated at room temperature, the extraction time is short, the total extraction time of single sample RNA is not longer than 10min (including gDNA removal), and the extraction efficiency is at least 50% higher than that of the existing RNA extraction kit;
2. The safety is high, reagents such as phenol, chloroform and the like are not required, ethanol precipitation is not required, each component formula does not relate to any toxic reagent, any toxic reagent is not required to be additionally added in the using process, and the whole extraction process is green, safe and nontoxic;
3. The application range is wide, and the method is applicable to RNA extraction of cells (such as mouse embryonic myoblast C2C12 and rat myocardial cell H9C 2), animal tissues (such as mouse liver tissue) and plant tissues (such as plant leaves);
4. the extraction purity is high, the gDNA and protein pollution is extremely low after the DNase and proteinase K are treated and then are subjected to column adsorption and washing for a plurality of times, the difference between the extracted RNA samples is small, and the quality is stable.
Drawings
FIG. 1 is a 1% agarose gel electrophoretogram of RNA. Wherein band M is RNA MARKER, bands 1-3 are electrophoresis bands of RNA extracted from mouse colorectal cancer cells of example 1; lanes 4-6 are electrophoresis lanes of RNA extracted from mouse liver tissue of example 2; lanes 7-9 are the electrophoresis lanes of RNA extracted from Arabidopsis thaliana leaves of example 3; lanes 10-12 are the electrophoresis lanes of RNA extracted from mouse colorectal cancer cells of comparative example 1. Sample loading amount: 2. Mu.L; electrophoresis conditions: 180V,8min.
Detailed Description
For a more specific understanding of the technical content, features and effects of the present invention, the technical solution of the present invention will be described in further detail with reference to the accompanying drawings and specific examples.
Example 1
1. Kit for detecting a substance in a sample
The rapid RNA extraction kit of the embodiment comprises a lysate, protease K, DNA enzyme, 10×reaction Buffer, a rinsing solution, an eluent, an RNA adsorption column and a collection tube. Wherein:
The lysate consisted of 4.0M guanidine isothiocyanate, 0.1M Tris-HCl pH5.0, 0.5% N-sarcosyl, 0.5M sodium chloride, 0.2% sucrose fatty acid ester, 0.2% Triton X-100, 40% isopropanol. Since isopropanol contains more hydroxyl groups, water can be mixed with salt ions, so that the isopropanol is added after other components are completely dissolved, the solution is clear and transparent, and otherwise, part of solute is separated out.
The concentration of proteinase K was 10mg/mL, >30U/mg.
The dnase is a double-strand specific dnase, and can specifically digest double-strand DNA without affecting single-strand DNA. DNase concentration was 3U/. Mu.L.
10 Xreaction Buffer consisted of 0.02M Tris-HCl, 0.02M magnesium sulfate, 3.0mM calcium chloride, pH 8.0.
The rinse solution consisted of 0.1M Tris-HCl pH7.0, 0.1M sodium chloride, 60% absolute ethanol (absolute ethanol added after other ingredients were completely dissolved).
The eluent was sterile ultrapure water from which rnase had been removed.
The RNA adsorption column is a silicon membrane adsorption column.
RNA extraction
This example uses, as a sample, mouse colorectal cancer adherent cells that were shed by pancreatin digestion. The method for extracting the sample RNA by using the kit in the step 1 comprises the following steps:
1) Lysing the samples: the detached adherent cells were collected by centrifugation at 6000rpm for 1min, 500. Mu.L of lysate and 50. Mu.L of proteinase K were added to the cells, and the mixture was vortexed and mixed for 30s, and allowed to stand at room temperature for 1min for reaction.
2) Rinsing impurities: 500. Mu.L of a rinse solution was added to the RNA adsorption column, the column was rinsed twice, then was air-separated (12000 rpm,30 s), and the column was allowed to stand for 1min after opening the lid to completely remove the rinse solution.
3) Eluting RNA: suspending and dripping 50 mu L of eluent on a silicon membrane of an RNA adsorption column, standing for 1min at room temperature, centrifuging (12000 rpm,1 min), and collecting filtrate to obtain RNA.
RNA extraction results
Three biological repeated experiments were performed on the adherent cell RNA extraction according to the above method, wherein each extraction process took 5 minutes, the concentration of RNA extraction was 654.21 ng/. Mu.L, 684.12 ng/. Mu.L, 664.87 ng/. Mu.L, and the ratio of A260/A280 was 2.01, 2.02, 2.04, and the ratio of A260/A230 was 2.04, 2.05, 2.04, respectively, as shown in Table 1.
TABLE 1 RNA extraction results of example 1
| Parallel example 1 | Parallel example 2 | Parallel example 3 | |
| A260/A280 | 2.01 | 2.02 | 2.04 |
| A260/A230 | 2.04 | 2.05 | 2.04 |
| Concentration (ng/. Mu.L) | 654.21 | 684.12 | 664.87 |
Example 2
1. Kit for detecting a substance in a sample
As in example 1.
RNA extraction
The present example uses mouse liver tissue as a sample. The method for extracting the sample RNA by using the kit in the step 1 is as follows:
1) Lysing the samples: grinding the tissue sample into solution by using a grinding rod, adding 500 mu L of lysate and 50 mu L of proteinase K into 20 mu L of tissue solution, vortex shaking and mixing for 30s, standing for 1min at room temperature for reaction, centrifuging (12000 rpm,1 min), and taking supernatant for subsequent extraction operation. Subsequent steps 2) to 3) are the same as in example 1.
RNA extraction results
Three biological repeated experiments were performed on RNA extraction from liver tissue of mice in the above manner, each time the extraction process took 5 minutes, the concentration of RNA extraction was 789.54 ng/. Mu.L, 788.52 ng/. Mu.L, 796.52 ng/. Mu.L, the ratio of A260/A280 was 2.05, 2.06, 2.08, and the ratio of A260/A230 was 2.08, 2.06, respectively, as shown in Table 2.
TABLE 2 RNA extraction results of example 2
| Parallel example 1 | Parallel example 2 | Parallel example 3 | |
| A260/A280 | 2.05 | 2.06 | 2.08 |
| A260/A230 | 2.08 | 2.06 | 2.06 |
| Concentration (ng/. Mu.L) | 789.54 | 788.52 | 796.52 |
Example 3
1. Kit for detecting a substance in a sample
The rapid RNA extraction kit of the embodiment comprises a lysate, protease K, DNA enzyme, 10×reaction Buffer, a rinse solution, an eluent, an RNA adsorption column and a collection tube. Wherein:
The lysate consisted of 6.0M guanidine isothiocyanate, 0.15M Tris-HCl pH6.5, 2.0% N-sarcosyl, 1.0M sodium chloride, 0.5% sucrose fatty acid ester, 1.0% Triton X-100, 50% isopropanol.
The concentration of proteinase K was 30mg/mL, >30U/mg.
The dnase is a double-strand specific dnase, and can specifically digest double-strand DNA without affecting single-strand DNA. DNase concentration was 3U/. Mu.L.
10 Xreaction Buffer consisted of 0.08M Tris-HCl, 0.02M magnesium sulfate, 3.0mM calcium chloride, pH 8.0.
The rinse solution consisted of 0.2M Tris-HCl, 0.2M sodium chloride, 80% absolute ethanol, pH 8.0.
The eluent was sterile ultrapure water from which rnase had been removed.
The RNA adsorption column is a silicon membrane adsorption column.
RNA extraction
The present example uses Arabidopsis leaves as a sample. The method for extracting sample RNA by using the kit of the step 1 in the embodiment is as follows:
1) Lysing the samples: the arabidopsis leaves are placed in liquid nitrogen for quick freezing, then grinding and crushing are carried out, about 100mg of sample is taken, 500 mu L of lysate and 50 mu L of proteinase K are added, vortex oscillation and mixing are carried out for 30s, standing is carried out for 1min at room temperature for reaction, and supernatant is taken after centrifugation (12000 rpm,1 min) for subsequent extraction operation.
Subsequent steps 2) to 3) are the same as in example 1.
RNA extraction results
Three biological replicates were performed on Arabidopsis leaf RNA extraction using the above method, each time the extraction process took 5 minutes, the concentration of RNA extraction was 542.21 ng/. Mu.L, 554.69 ng/. Mu.L, 585.41 ng/. Mu.L, the ratio of A260/A280 was 2.05, 2.04, the ratio of A260/A230 was 2.11, 2.08, respectively, as shown in Table 3.
TABLE 3 RNA extraction results from example 3
| Parallel example 1 | Parallel example 2 | Parallel example 3 | |
| A260/A280 | 2.05 | 2.05 | 2.04 |
| A260/A230 | 2.11 | 2.11 | 2.08 |
| Concentration (ng/. Mu.L) | 542.21 | 554.69 | 585.41 |
Comparative example 1
1. Kit for detecting a substance in a sample
The comparative example used the conventional TIANGEN RNASIMPLE total RNA extraction kit (DP 419) which included: lysate, deproteinized solution, rinse solution, RNase-Free double distilled water, RNase-Free adsorption column, and RNase-Free centrifuge tube.
RNA extraction
The present comparative example uses, as a sample, mouse colorectal cancer adherent cells that were shed by pancreatin digestion. The method for extracting total RNA of the sample by using the kit in the step 1 of the comparative example is as follows:
1) Sample treatment: cells were harvested by centrifugation and the supernatant discarded. 1mL of lysate was added to each 5-10X 106 cells, and the cells were not washed before addition of lysate, so as not to degrade mRNA.
2) 200. Mu.L of chloroform was added thereto, the tube was covered with a cap, vigorously shaken for 15sec, and left at room temperature for 3min.
3) Centrifugation (4 ℃,12000rpm,10 min), the sample will be divided into three layers: a yellow organic phase, an intermediate layer and a colorless aqueous phase, the RNA being predominantly in the aqueous phase, the volume of the aqueous phase being about 50% of the lysate reagent used. The aqueous phase was transferred to a new tube for the next operation.
4) Slowly adding 0.5 times volume of absolute ethyl alcohol, mixing uniformly (precipitation possibly occurs at this time), transferring the obtained solution and the precipitate into an RNase-Free adsorption column, centrifuging at 12000rpm for 30sec at 4 ℃, if the whole solution and the mixture cannot be added into the RNase-Free adsorption column at one time, transferring into the RNase-Free adsorption column twice, centrifuging at 12000rpm for 30sec at 12000rpm at 4 ℃, and discarding the waste liquid in the collecting pipe.
5) 500. Mu.L deproteinized solution (before use, it was checked whether ethanol had been added) was added to the RNase-Free adsorption column, and the column was centrifuged at 12000rpm at 4℃for 30sec, and the waste solution was discarded, and the RNase-Free adsorption column was placed in a collection tube.
6) 500. Mu.L of the rinse solution (please check whether ethanol has been added) was added to the RNase-Free adsorption column, and the mixture was allowed to stand at room temperature for 2min, at 4℃and centrifuged at 12000rpm for 30sec, and the waste solution was discarded.
7) Repeating the operation step 6).
8) The RNase-Free adsorption column was placed in a 2ml collection tube, centrifuged at 12000rpm at 4℃for 2min, and the residual liquid was removed. After centrifugation, the RNase-Free adsorption column is placed at room temperature for a moment or placed on an ultra-clean workbench for ventilation for a moment to be fully dried.
9) Transferring the RNase-Free adsorption column into a new 1.5ml centrifuge tube, adding 30-100 mu L of RNase-Free ddH 2 O, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min at the temperature of 4 ℃ to obtain RNA.
RNA extraction results
Three biological replicates were performed for adherent cell RNA extraction as described above, each time taking 27min, the concentration of RNA extraction was 421.52 ng/. Mu.L, 408.32 ng/. Mu.L, 412.62 ng/. Mu.L, the ratio A260/A280 was 1.87, 1.88, 1.81, the ratio A260/A230 was 1.94, 1.91, 1.89, respectively, as shown in Table 4.
TABLE 4 RNA extraction results of comparative example 1
| Parallel example 1 | Parallel example 2 | Parallel example 3 | |
| A260/A280 | 1.87 | 1.88 | 1.81 |
| A260/A230 | 1.94 | 1.91 | 1.89 |
| Concentration (ng/. Mu.L) | 421.52 | 408.32 | 412.62 |
RNA extraction effects of examples 1 to 3 and comparative example 1 referring to the RNA gel electropherograms of FIG. 1, the clarity and brightness of 28s, 18s, 5s bands were observed.
As can be seen from the RNA extraction results of examples 1 to 3 and comparative example 1, the concentration and purity of RNA extracted in examples 1 to 3 are both superior to those of comparative example 1, and the concentration of RNA extracted in example 2 is significantly higher than those of examples 1 and 3, indicating that the kit of the present invention is particularly suitable for extraction of RNA from animal tissues. More importantly, the time for extracting RNA in examples 1-3 is about 5min, while the time for extracting RNA in comparative example 1 is about 30min, so that the experimental time can be greatly saved by using the kit provided by the invention.
The foregoing embodiments are merely examples of possible or preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and therefore, all equivalent changes and modifications that are consistent with the scope of the present invention shall fall within the scope of the present invention.
Claims (14)
- The rapid extraction kit for RNA is characterized in that: comprises a lysate, proteinase K, a rinsing liquid, an eluent, an RNA adsorption column and a collecting pipe, wherein the lysate comprises guanidine isothiocyanate, tris-HCl, N-dodecyl sodium sarcosinate, sodium chloride, sucrose fatty acid ester, triton X-100 and isopropanol.
- 2. The kit of claim 1, further comprising dnase and a reaction buffer.
- 3. The kit according to claim 2, wherein the DNase is a double-strand specific DNase, the Reaction Buffer is 10 Xreaction Buffer, and the kit comprises 0.02-0.08M Tris-HCl with pH of 8.0, 0.01-0.02M magnesium sulfate and 1.0-3.0 mM calcium chloride.
- 4. The kit according to claim 1, wherein the lysate contains guanidine isothiocyanate 4.0-8.0M, tris-HCl 0.02-0.15M at ph5.0-6.5, N-dodecyl sodium sarcosinate 0.2-2.0% by mass, sodium chloride 0.2-1.0M, sucrose fatty acid ester 0.1-0.5% by mass, triton X-100 0% by volume, and isopropyl alcohol 30-50% by volume.
- 5. The kit according to claim 4, wherein the lysate contains 4.0M guanidine isothiocyanate, 0.1M Tris-HCl having pH of 5.0, 0.5% N-dodecylsarcosine sodium, 0.5M sodium chloride, 0.2% sucrose fatty acid ester, 0.2% Triton X-100 by volume, and 40% isopropyl alcohol by volume.
- 6. The kit according to claim 4, wherein the lysate contains 6.0M guanidine isothiocyanate, 0.15M Tris-HCl having pH6.5, 2.0% N-dodecylsarcosine sodium, 1.0M sodium chloride, 0.5% sucrose fatty acid ester, 1.0% Triton X-100 by volume, 50% isopropyl alcohol by volume.
- 7. The kit of claim 1, wherein the concentration of proteinase K is 10mg/mL to 30mg/mL, >30U/mg.
- 8. The kit according to claim 1, wherein the rinsing solution contains Tris-HCl 0.02-0.2M, sodium chloride 0.02-0.2M, and absolute ethanol with a volume fraction of 60% -80% at ph 6.0-8.0.
- 9. The kit of claim 8, wherein the rinse solution comprises 0.1M Tris-HCl pH7.0, 0.1M sodium chloride, 60% absolute ethanol.
- 10. The kit of claim 8, wherein the rinse solution comprises 0.2M Tris-HCl, 0.2M sodium chloride, 80% absolute ethanol at ph 8.0.
- 11. The kit according to claim 1, wherein the eluent is sterilized ultrapure water from which rnase is removed, and the RNA adsorption column is a silicon membrane adsorption column.
- 12. Use of a kit according to any one of claims 1-11 for rapid extraction of RNA.
- 13. A method for rapid extraction of RNA using the kit of any one of claims 1 to 11, comprising the steps of:1) Lysing the samples: adding lysate and proteinase K into the treated cells or tissues, mixing, and standing at room temperature for reaction;2) Rinsing impurities: adding a rinsing liquid into the RNA adsorption column, performing air-separation after rinsing, uncovering and standing, and thoroughly removing the rinsing liquid;3) Eluting RNA: suspending and dripping eluent on the RNA adsorption column, standing at room temperature, centrifuging and collecting filtrate to obtain RNA.
- 14. The method according to claim 13, further comprising the steps between step 1) and step 2) of:Removing gDNA: transferring the cracking mixture obtained after the reaction into an RNA adsorption column, centrifuging, suspending and dripping a DNase working solution prepared from DNase and a reaction buffer solution on the RNA adsorption column, and reacting at room temperature.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410462002.6A CN118165982A (en) | 2024-04-17 | 2024-04-17 | Kit for rapidly extracting RNA and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410462002.6A CN118165982A (en) | 2024-04-17 | 2024-04-17 | Kit for rapidly extracting RNA and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118165982A true CN118165982A (en) | 2024-06-11 |
Family
ID=91347030
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410462002.6A Pending CN118165982A (en) | 2024-04-17 | 2024-04-17 | Kit for rapidly extracting RNA and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118165982A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130344480A1 (en) * | 2012-06-20 | 2013-12-26 | Arkray, Inc. | Method for Treating a Blood Component Containing Sample |
| CN107922971A (en) * | 2015-05-18 | 2018-04-17 | 凯锐思公司 | Composition and method for enriched nucleic acid colony |
| CN111961664A (en) * | 2020-09-08 | 2020-11-20 | 武汉中投汉嘉科技有限公司 | Nucleic acid extracting solution |
| CN112725334A (en) * | 2021-02-23 | 2021-04-30 | 山东思科捷生物技术有限公司 | Cell RNA rapid extraction kit and RNA extraction method |
| CN117210454A (en) * | 2023-11-09 | 2023-12-12 | 北京纳捷诊断试剂有限公司 | Lysate for extracting nucleic acid from whole blood sample and application thereof |
-
2024
- 2024-04-17 CN CN202410462002.6A patent/CN118165982A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130344480A1 (en) * | 2012-06-20 | 2013-12-26 | Arkray, Inc. | Method for Treating a Blood Component Containing Sample |
| CN107922971A (en) * | 2015-05-18 | 2018-04-17 | 凯锐思公司 | Composition and method for enriched nucleic acid colony |
| CN111961664A (en) * | 2020-09-08 | 2020-11-20 | 武汉中投汉嘉科技有限公司 | Nucleic acid extracting solution |
| CN112725334A (en) * | 2021-02-23 | 2021-04-30 | 山东思科捷生物技术有限公司 | Cell RNA rapid extraction kit and RNA extraction method |
| CN117210454A (en) * | 2023-11-09 | 2023-12-12 | 北京纳捷诊断试剂有限公司 | Lysate for extracting nucleic acid from whole blood sample and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101218469B1 (en) | Method for separating purified RNA | |
| EP1994142B1 (en) | Methods and compositions for the rapid isolation of small rna molecules | |
| US6762027B2 (en) | Compositions, methods, and kits for isolating nucleic acids using surfactants and proteases | |
| EP2580323B1 (en) | Extraction of nucleic acids from wax-embedded samples | |
| KR101641113B1 (en) | Solution for extraction of | |
| AU2001297835A1 (en) | Compositions, methods, and kits for isolating nucleic acids using surfactants and proteases | |
| CN116334071A (en) | Nucleic acid extraction kit for detecting pathogenic microorganisms and extraction method thereof | |
| Bharudin et al. | COMPARISON OF RNA EXTRACTION METHODS FOR TRANSCRIPT ANALYSIS FROM THE PSYCHROPHILIC YEAST, Glaciozyma antarctica. | |
| CN101591650B (en) | A reagent for isolating RNA from biological tissue, cells, or blood samples | |
| CN112725334B (en) | Cell RNA rapid extraction kit and RNA extraction method | |
| CN118165982A (en) | Kit for rapidly extracting RNA and application thereof | |
| CN108676795B (en) | Non-toxic extract solution combination GNR.1 for efficient extraction of plant genomic DNA and extraction method | |
| Ahmed et al. | Low-cost and highly efficient: A method for high-quality nucleic acid isolation from cotton fibres | |
| RU2672378C1 (en) | Method for dna isolation from plants, suitable for pcr | |
| Sreeja et al. | A modified protocol for isolation of high quality total RNA from ginger (Zingiber officinale Rosc.) rhizomes | |
| Thomas et al. | Improved method for purification of RNA from stem tissue of grapevine and its use in mRNA profiling | |
| KR101620481B1 (en) | A Efficient Method for Extracting DNA from Eukaryote Algae | |
| EP2196537A1 (en) | Method for synthesis of single- or double-stranded dna, and kit for the synthesis | |
| WO2022139709A1 (en) | Modified method for high quality and pure rna isolation from pomegranate | |
| CN120330294A (en) | Extraction method and application of high molecular weight vortex worm genome DNA | |
| CN118638776A (en) | Composition for removing inhibitors from samples, method for separating nucleic acids, kit and application thereof | |
| CN115161315A (en) | Plant RNA Rapid Extraction Kit | |
| Parker | The Relative Recoverability of Dna And Rna Profiles from Forensically Relevant Body Fluid Stains |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |