CN116590282A - Kit for extracting total RNA in whole blood and method thereof - Google Patents
Kit for extracting total RNA in whole blood and method thereof Download PDFInfo
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- CN116590282A CN116590282A CN202310750387.1A CN202310750387A CN116590282A CN 116590282 A CN116590282 A CN 116590282A CN 202310750387 A CN202310750387 A CN 202310750387A CN 116590282 A CN116590282 A CN 116590282A
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- 238000004140 cleaning Methods 0.000 claims abstract description 36
- 239000007788 liquid Substances 0.000 claims abstract description 35
- 239000000243 solution Substances 0.000 claims abstract description 31
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- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims abstract description 19
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 16
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 12
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000005336 cracking Methods 0.000 claims abstract description 10
- 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 8
- 229960000789 guanidine hydrochloride Drugs 0.000 claims abstract description 4
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims abstract description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 4
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- 239000011324 bead Substances 0.000 claims description 21
- 238000007885 magnetic separation Methods 0.000 claims description 15
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- 239000006228 supernatant Substances 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 3
- 125000000896 monocarboxylic acid group Chemical group 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000008188 pellet Substances 0.000 claims description 3
- 238000000197 pyrolysis Methods 0.000 claims description 3
- 239000004005 microsphere Substances 0.000 abstract description 12
- 238000000605 extraction Methods 0.000 abstract description 11
- 210000003743 erythrocyte Anatomy 0.000 abstract description 9
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- 102000039446 nucleic acids Human genes 0.000 description 11
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- 150000007523 nucleic acids Chemical class 0.000 description 11
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- 108090000623 proteins and genes Proteins 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
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- 238000002474 experimental method Methods 0.000 description 5
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- 102000016911 Deoxyribonucleases Human genes 0.000 description 4
- 108010053770 Deoxyribonucleases Proteins 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
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- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
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- 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
- C12N15/1013—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 by using magnetic beads
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Abstract
The invention discloses a kit for extracting total RNA in whole blood and a method thereof, wherein the kit comprises a lysate A, a lysate B, a lysate C, a cleaning solution A and a cleaning solution B, wherein the kit comprises the following components: the cracking liquid A is a mixed liquid composed of 2-4.5M guanidine isothiocyanate, 5-25% of triton, 0.01-0.05M hydrochloric acid, 0.1-0.2M ethylenediamine tetraacetic acid and 1-2M tris (hydroxymethyl) aminomethane hydrochloride, the cracking liquid B is a mixed liquid composed of 2-4.5M guanidine isothiocyanate, 5-25% of triton, 0.01-0.1M sodium hydroxide, 0.1-0.2M ethylenediamine tetraacetic acid and 1-2M tris (hydroxymethyl) aminomethane, the cracking liquid C is a mixed liquid composed of 2-4.5M guanidine isothiocyanate, 10-30% triton, 0.01-0.1M hydrochloric acid, 0.1-0.2M ethylenediamine tetraacetic acid and 1-2M tris (hydroxymethyl) aminomethane hydrochloride, the cracking liquid A is a mixed liquid composed of 2-5M guanidine hydrochloride, 50-70% ethanol and 10-30% triton, and the cracking liquid C is a mixed liquid composed of 2-4.5% ethanol and 75% ethanol. The invention also provides a method for extracting total RNA from whole blood by using the kit. The invention can extract total RNA in whole blood without erythrocyte lysis, and has simpler operation. The collocation of the kit can improve the sample processing and loading range of the extraction kit, and can prevent the agglomeration phenomenon of the magnetic microspheres caused by excessive adjustment to the greatest extent.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit for extracting total RNA in whole blood and a method thereof.
Background
The current method for extracting total RNA (ribonucleic acid) from blood generally comprises separating white blood cells from blood after erythrocyte lysis, and then performing cell lysis and nucleic acid purification. The extraction method of RNA commonly used at present is phenol-chloroform extraction method, but the defects are: 1. requiring specialized training personnel to operate; 2. at least 1ml of normal human blood is required to ensure success. After the advancement of material science, it has evolved: the reagent boxes of the two methods are portable reagent boxes at present and cannot be compatible with an automatic extraction process, and the maximum blood sample amount is 1.5ml of normal human whole blood, so that the conditions of poor purity and poor fragments of sample extraction can be caused in operation.
Disclosure of Invention
In order to solve the technical problems in the background technology, the invention provides a kit for extracting total RNA in whole blood and a method thereof.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a kit for extracting total RNA in whole blood comprises a lysate A, a lysate B, a lysate C, a cleaning solution A and a cleaning solution B, wherein:
the lysate A is a mixed solution composed of 2-4.5M guanidine isothiocyanate, 5-25% of triton, 0.01-0.05M hydrochloric acid, 0.1-0.2M ethylenediamine tetraacetic acid and 1-2M tris (hydroxymethyl) aminomethane hydrochloride,
the pyrolysis liquid B is a mixed liquid composed of 2-4.5M guanidine isothiocyanate, 5-25% of triton, 0.01-0.1M sodium hydroxide, 0.1-0.2M ethylenediamine tetraacetic acid and 1-2M tris (hydroxymethyl) aminomethane,
the lysate C is a mixed solution composed of 2-4.5M guanidine isothiocyanate, 10-30% of triton, 0.01-0.1M hydrochloric acid, 0.1-0.2M ethylenediamine tetraacetic acid and 1-2M tris (hydroxymethyl) aminomethane hydrochloride,
the cleaning solution A is a mixed solution composed of 2-5M guanidine hydrochloride, 50-70% ethanol and 10-30% triton,
the cleaning liquid B is a mixed liquid composed of 75-90% of ethanol and 5-30% of triton.
Further, the pH value of the lysate A is 3-6.
Further, the pH of the lysate B is 8-11.
Further, the pH of the lysate C is 4-7.
A method for extracting total RNA in whole blood comprises the following steps:
s1, taking a clean enzyme-free tube, adding the whole blood ratio liquid into the lysate B according to claim 1 in a mode of 1:1.5-1.6, and mixing for 5-10 min at 15-37 ℃;
s2, adding the cracking liquid A of claim 1 in a ratio of 1:0.6 into the mixed liquid in the step S1, and mixing for 5-10 min at 15-37 ℃;
s3, adding magnetic pellets with COOH or OH groups and with the particle size of 150-500 nm into the mixed solution in the step S2, and mixing for 5-10 min at 15-37 ℃;
s4, placing the mixture obtained in the step S3 on the side of a magnet or a commercial magnet holder, magnetically separating, and removing supernatant;
s5, placing the magnetic beads separated in the step S4 into the lysate C of claim 1, and performing magnetic separation after mixing for 5-10 min;
s6, placing the magnetic beads separated in the step S5 in a cleaning solution A for magnetic mixing and dispersing, and then performing magnetic separation;
s7, placing the magnetic beads separated in the step S6 in a cleaning solution B for magnetic mixing and dispersing, and then performing magnetic separation;
s8, placing the magnetic beads separated in the step S7 in a cleaning solution A for magnetic mixing and dispersing, and then performing magnetic separation;
s9, placing the magnetic beads separated in the step S8 in a cleaning solution B for magnetic mixing and dispersing, and then performing magnetic separation;
s10, mixing the magnetic beads separated in the step S9 with 50-200 u of enzyme-free water, incubating for 10-15 min at 45-60 ℃ and mixing every 5min to disperse the magnetic beads.
The applicant found that when the total RNA was extracted and purified from a blood sample and a bone-associated blood sample of a leukemia patient, the blood and bone-associated blood sample of the leukemia patient contained immature blastocyst cells, and the blood sample of the blood sample was different from that of a normal human sample in that the blood sample contained more useless leukemia cells (blood cancer cells). Therefore, when the cells are lysed, more gel substances are released due to the larger content of the whole cells, so that the recovery of the magnetic microspheres for nucleic acid recovery is interfered by the gel substances. In order to decompose such gel substances, the applicant has repeatedly studied and found that increasing the ph value can destroy the structure of the decomposed gel substances after trying a plurality of methods, but since the structural characteristics of RNA molecules cannot exist in environments with high ph values, the RNA molecules need to be immediately neutralized with acid and alkali to be protected, and thus the applicant has set lysates with different ph values to realize protection of RNA molecules and decomposition of gel substances. Because leukemia patients contain more immature bud ball cells in blood, the structures of gel substances are required to be broken down under the environment of higher pH value, so that magnetic microspheres are placed in the environment of higher nucleic acid and protein content to act, and considering that the magnetic microspheres can bind protein and nucleic acid simultaneously, the problem of protein cleaning is considered to purify nucleic acid samples with higher quality, and therefore, the applicant selects different neutral cleaning solutions.
Meanwhile, in the process of combining nucleic acid by the magnetic microspheres, the magnetic microspheres adsorb not only nucleic acid but also protein and salt, so that the quality and purity of a nucleic acid solution can be ensured only by cleaning the protein and removing the salt by a cleaning solution, in addition, genomic DNA is usually polluted when RNA is extracted, DNase nuclease is required to degrade genomic DNA, but DNase is protein and is easy to interfere with activity by salt substances, so that the applicant designs the cleaning solution (A, B), firstly cleans the salt adsorbed in a sample lysate, then uses DNase to act with the magnetic microspheres but has nucleic acid, and then mixes the adsorbed protein and salt with the cleaning solution (C, D) for cleaning and enzyme action.
The principle of extracting total RNA in whole blood is as follows: because RNA is easier to be acted by RNase nucleic acid degrading enzyme, chemical substances with strong protein activity inhibiting capability are needed to be used for extracting total RNA of whole blood, red blood cells are selectively cracked by using a red blood cell lysate at present, white blood cells are rapidly cracked by using unique lysate/beta-mercaptoethanol, cellular RNase is inactivated, then RNA is selectively adsorbed on a silicon matrix membrane in a centrifugal column in a high-order salt state after the combination condition is regulated by using ethanol, then cell metabolites, proteins and other impurities are removed by using a series of rapid rinsing-centrifuging steps, and finally pure RNA is eluted from magnetic microspheres by using low-salt RNase free H20. The method provides a unique lysate which can decompose protein gel substances, protect RNA integrity and prevent red blood cells from cracking, deteriorating and agglomerating, interfere the combination of magnetic microspheres and nucleic acid molecules, and elute RNA molecules by mixing the magnetic microspheres with enzyme-free water after cleaning the magnetic microspheres by protein cleaning liquid and salt ion removing liquid with different components.
Compared with the prior art, the invention has the beneficial effects that:
1. the method can extract total RNA in whole blood without erythrocyte lysis, and is simpler and more convenient to operate.
2. The matching of the reagent kit lysate A and the lysate B can improve the sample processing and loading range of the sample of the extraction reagent kit.
3. The combination of the kit lysate and the cleaning liquid can prevent the magnetic microspheres from agglomerating due to excessive adjustment to the greatest extent.
4. The kit can be manually operated or matched with a Concertbio HF serial pipetting automatic extractor, so that the whole blood can be directly put on the machine to extract the total RNA in the whole blood.
5. A one-time sampling extraction may be achieved.
6. The kit can purify 3ug total RNA in 1ml blood, and has high extraction efficiency.
Drawings
The invention will be further described with reference to examples of embodiments with reference to the accompanying drawings.
FIG. 1 is a 1.2% agarose gel (110V 10 min) of total RNA extraction yield evaluation of whole blood before formulation adjustment optimization.
FIG. 2 is a graph of 1.2% agarose gel (110V 10 min) of total RNA extraction yield evaluation of whole blood after formula adjustment optimization.
FIG. 3 is a graph of 1.2% agarose gel (110V 10 min) of total RNA extraction yield from whole blood using lysate A alone.
Detailed Description
For a better understanding of the present invention, reference will now be made in detail to the following examples and accompanying drawings, which are included to provide a further understanding of the invention, and it is to be understood by those skilled in the art that the following examples are not intended to limit the scope of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 kit composition
A kit for extracting total RNA in whole blood comprises a lysate A, a lysate B, a lysate C, a cleaning solution A and a cleaning solution B, wherein:
the lysate A is a mixed solution composed of 2-4.5M guanidine isothiocyanate, 5-25% of triton, 0.01-0.05M hydrochloric acid, 0.1-0.2M ethylenediamine tetraacetic acid and 1-2M tris (hydroxymethyl) aminomethane hydrochloride, and the PH value is 3-6.
The lysate B is a mixed solution composed of 2-4.5M guanidine isothiocyanate, 5-25% of triton, 0.01-0.1M sodium hydroxide, 0.1-0.2M ethylenediamine tetraacetic acid and 1-2M tris (hydroxymethyl) aminomethane, and the PH value is 9.5-11.
The lysate C is a mixed solution composed of 2-4.5M guanidine isothiocyanate, 10-30% of triton, 0.01-0.1M hydrochloric acid, 0.1-0.2M ethylenediamine tetraacetic acid and 1-2M tris (hydroxymethyl) aminomethane hydrochloride, and the PH value is 4-7.
The cleaning solution A is a mixed solution composed of 2-5M guanidine hydrochloride, 50-70% ethanol and 10-30% triton,
the cleaning liquid B is a mixed liquid composed of 75-90% of ethanol and 5-30% of triton.
Example 2 method for extracting Total RNA from Whole blood
A method for extracting total RNA in whole blood comprises the following steps:
s1, taking a clean 5 ml-15 ml enzyme-free tube, adding the whole blood ratio liquid into the lysis solution B in the embodiment 1 in a mode of 1:1.5-1.6, and mixing for 5-10 min at 15-37 ℃;
s2, adding the cracking solution A in the embodiment 1 with the ratio of 1:0.6 into the mixed solution in the step S1, and mixing for 5-10 min at 15-37 ℃;
s3, adding magnetic pellets with COOH or OH groups and with the particle size of 150-500 nm into the mixed solution in the step S2, and mixing for 5-10 min at 15-37 ℃;
s4, placing the mixture obtained in the step S3 on the side of a magnet or a commercial magnet holder, magnetically separating, and removing supernatant;
s5, placing the magnetic beads separated in the step S4 into the pyrolysis liquid C of the embodiment 1, and carrying out magnetic separation after mixing for 5-10 min;
s6, placing the magnetic beads separated in the step S5 in a cleaning solution A for magnetic mixing and dispersing, and then performing magnetic separation;
s7, placing the magnetic beads separated in the step S6 in a cleaning solution B for magnetic mixing and dispersing, and then performing magnetic separation;
s8, placing the magnetic beads separated in the step S7 in a cleaning solution A for magnetic mixing and dispersing, and then performing magnetic separation;
s9, placing the magnetic beads separated in the step S8 in a cleaning solution B for magnetic mixing and dispersing, and then performing magnetic separation;
s10, mixing the magnetic beads separated in the step S9 with 50-200 u of enzyme-free water, incubating for 10-15 min at 45-60 ℃ and mixing every 5min to disperse the magnetic beads.
Blood cell concentration operation steps:
the purpose of the experiment was to simulate a blood sample with a high cell content, 1000, 2000, 3000, 4000 blood samples were concentrated to 400ul.
Sample processing:
this experiment required 20.8ml of fresh blood.
16.8ml of blood was taken and 989ul of each tube was dispensed into 17 5ml centrifuge tubes, each with 3ml of RBC lysis buffer. After being mixed by gentle inversion, the mixture is placed at 4 ℃ for 10min, and then centrifuged at 3000rpm for 10min, the centrifugal sediment is collected and then dissolved back to 1680ul by RBC lysis buffer solution, 60ul, 160 ul, 260 ul and 360ul of the solution are respectively taken out of a brand new 5ml centrifuge tube, two parallel centrifuge tubes are taken out for 8 parts per volume, and 1000, 2000, 3000 and 4000 are marked in sequence respectively.
The RBC lysis buffer was added to 4ml of the 8 centrifuge tubes filled with leukocytes from the previous step, gently mixed with a pipette, centrifuged on a horse, and centrifuged at 12000rpm for 3min. After centrifugation, the supernatant was discarded, white blood cells were reconstituted with 400ul of fresh blood per tube, the treated samples were transferred to sample tubes, and 40ul (20 mg/ml) proteinase K was added. Two 400ul whole blood aliquots were prepared and 40ul (20 mg/ml) proteinase K was added as well. The experimental procedure was performed with a 400ul system sample processing system, nucleic acid elution volume of 60ul.
Intervention in Dnase1 treatment during the experiment: 1600ul DNase/RNase-free-water was taken into a 5ml centrifuge tube, and 200ul DNase1, 200ul 10X Reaction Buffer was added simultaneously. And after vortex mixing, 200ul of the mixture is taken and split into large heating tanks for 10 tests. The total RNA extraction yield of whole blood before the optimization of the formulation was not evaluated, and the results are shown in Table 1 and FIG. 1.
Table 1 evaluation Table of total RNA extraction yield from Whole blood before optimization of formulation adjustment
The pH of the lysate B was set to 8-11, the pH of the lysate A was set to 4-6, and the total RNA extraction yield of whole blood after the formulation was adjusted and optimized was evaluated in the same manner as described above, and the results are shown in Table 2 and FIG. 2.
Table 2 evaluation of total RNA extraction yield from whole blood after formulation adjustment and optimization
The results of the same procedure as described above except that only lysate A having a pH of 4-6 was used are shown in Table 3 and FIG. 3. As a result, it was found that only 400 to 2000. Mu.l of whole blood sample white blood cells could be processed by using only the lysate A having a pH of 4 to 6, and the extraction stability was lowered if the sample size was further enlarged.
TABLE 3 Total RNA extraction yields from Whole blood with lysate A alone
As is clear from the above experimental results, the molecular weight of RNA extracted from the treatment group of lysate B (pH 8-11) +lysate A (pH 4-6) was directly proportional to the amount of blood sample used, and it was found that the extraction stability and the throughput of blood cells were significantly improved as compared with the extraction group without adjustment or with the extraction group using a single component lysate A (pH 4-6).
While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that the specific embodiments described are illustrative only and not intended to limit the scope of the invention, and that equivalent modifications and variations of the invention in light of the spirit of the invention will be covered by the claims of the present invention.
Claims (5)
1. The kit for extracting total RNA in whole blood is characterized by comprising a lysate A, a lysate B, a lysate C, a cleaning solution A and a cleaning solution B, wherein:
the cracking liquid A is a mixed liquid composed of 2-4.5M guanidine isothiocyanate, 5-25% triton, 0.01-0.05M hydrochloric acid, 0.1-0.2M ethylenediamine tetraacetic acid and 1-2M tris (hydroxymethyl) aminomethane hydrochloride,
the pyrolysis liquid B is a mixed liquid composed of 2-4.5M guanidine isothiocyanate, 5-25% triton, 0.01-0.1M sodium hydroxide, 0.1-0.2M ethylenediamine tetraacetic acid and 1-2M tris (hydroxymethyl) aminomethane,
the lysate C is a mixed solution composed of 2-4.5M guanidine isothiocyanate, 10-30% of triton, 0.01-0.1M hydrochloric acid, 0.1-0.2M ethylenediamine tetraacetic acid and 1-2M tris (hydroxymethyl) aminomethane hydrochloride,
the cleaning liquid A is a mixed liquid composed of 2-5M guanidine hydrochloride, 50-70% ethanol and 10-30% triton,
the cleaning liquid B is a mixed liquid consisting of 75-90% of ethanol and 5-30% of triton.
2. The kit according to claim 1, wherein the pH of the lysate A is 3-6.
3. The kit according to claim 1, wherein the pH of the lysate B is 8-11.
4. The kit according to claim 1, wherein the pH of the lysate C is 4-7.
5. The method for extracting total RNA in whole blood is characterized by comprising the following steps:
s1, taking a clean enzyme-free tube, adding the whole blood ratio liquid into the lysate B according to claim 1 in a mode of 1:1.5-1.6, and mixing for 5-10 min at 15-37 ℃;
s2, adding the cracking liquid A of claim 1 in a ratio of 1:0.6 into the mixed liquid in the step S1, and mixing for 5-10 min at 15-37 ℃;
s3, adding magnetic pellets with COOH or OH groups and with the particle size of 150-500 nm into the mixed solution in the step S2, and mixing for 5-10 min at 15-37 ℃;
s4, placing the mixture obtained in the step S3 on the side of a magnet or a commercial magnet holder, magnetically separating, and removing supernatant;
s5, placing the magnetic beads separated in the step S4 into the lysate C of claim 1, and performing magnetic separation after mixing for 5-10 min;
s6, placing the magnetic beads separated in the step S5 in a cleaning solution A for magnetic mixing and dispersing, and then performing magnetic separation;
s7, placing the magnetic beads separated in the step S6 in a cleaning solution B for magnetic mixing and dispersing, and then performing magnetic separation;
s8, placing the magnetic beads separated in the step S7 in a cleaning solution A for magnetic mixing and dispersing, and then performing magnetic separation;
s9, placing the magnetic beads separated in the step S8 in a cleaning solution B for magnetic mixing and dispersing, and then performing magnetic separation;
s10, mixing the magnetic beads separated in the step S9 with 50-200 u of enzyme-free water, incubating for 10-15 min at 45-60 ℃ and mixing every 5min to disperse the magnetic beads.
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| CN202310750387.1A CN116590282A (en) | 2023-06-25 | 2023-06-25 | Kit for extracting total RNA in whole blood and method thereof |
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