CN111607592B - Method for extracting RNA (ribonucleic acid) in early development stage of cotton fiber cells - Google Patents
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Abstract
The technology belongs to the field of biotechnology, and discloses a method for extracting RNA (ribonucleic acid) in early development stage of cotton fiber cells, which comprises the steps of taking a plurality of ovaries in different development stages, and taking complete ovules; putting the ovule into lysis solution, vacuumizing, centrifuging, and taking supernatant; filtering the supernatant, adding absolute ethyl alcohol into the filtered supernatant to precipitate nucleic acid, collecting the obtained solution, adding deproteinizing solution RW1 to wash protein, adding DNase I working solution to digest DNA, adding deproteinizing solution RW1 and rinsing solution RW to wash other impurities except the nucleic acid, and collecting the eluted RNA solution; and finally, detecting the concentration and purity of the RNA. The invention is applicable to any cotton variety. Can quickly extract cotton fiber cell development early specific RNA which is not easy to separate in the periods of-1 DPA, 0DPA, 1DPA, 2DPA, 3DPA, 4DPA and 5DPA, and meets the requirement of a transcription and tissue library construction.
Description
Technical Field
The technology belongs to the technical field of biology, and particularly relates to a method for extracting RNA in early development stage of cotton fiber cells.
Background
Cotton is an important economic crop, is a main source of natural fiber, has great economic significance in textile production, and has important biological significance. Cotton fibers are single cells formed by ovule exodermal cells through special differentiation processes, and are a model system for studying cell elongation and cell wall synthesis. Cotton fiber development includes four stages: initiation of differentiation of fibroblasts, elongation of fibroblasts, thickening of secondary walls and maturation by dehydration. The initiation of fiber generally occurs from 3 days before flowering to 5 days after flowering, i.e., -3DPA to 5DPA (days post anthesis). The differentiation initiation of fiber cells is an extremely complex and crucial process, which not only determines the quantity and development condition of fibers, but also influences the quality and yield of cotton, and elucidating the fiber initiation mechanism has important significance for breeding new varieties of high-quality and high-yield cotton.
The current methods for researchers to obtain early stage cotton fiber tissue are mainly the following three.
(1) Analyzing the gene expression level of early-stage cotton fiber cells by taking the whole ovule in the early development stage as a research object. The biggest problem of the method is poor tissue specificity, because tissues such as an external integument, an internal integument and a nucellus are mixed in the intact ovule besides early cotton fiber cells, the specific analysis of the early development of cotton fibers is seriously interfered.
(2) Laser capture microdissection of fiber-initiating cells, i.e., obtaining fiber-initiating cells from early ovule sections using laser capture microdissection techniques. The method has high dependence on a technical platform, complicated procedures, time and labor waste and cross contamination.
(3) Ultra-low temperature mechanical shaking separation, namely putting the early ovule into liquid nitrogen, adding a glass bead, shaking, and collecting the exfoliated ovule epidermal cells. The method is unstable, has serious pollution and low yield of fiber cells.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) the RNA sample of the cells in the early development stage of cotton fiber is mainly composed of fiber cells and other parts of ovule except the fiber cells, the proportion of the fiber cells in the total cells is only less than 1 percent, and the research of the transcription level in the initial development stage of the fiber is severely limited.
(2) Although specific cotton fiber early-stage developmental cells can be obtained by using the laser microdissection technology, the total amount of the cells obtained by the method rarely needs to be amplified twice to meet the requirement of high-throughput library construction sequencing, and a system error is introduced in the continuous amplification process, so that the accuracy of a transcription level research result is reduced.
(3) Although the ultra-low temperature mechanical oscillation method can separate relatively specific cotton fiber early-stage development cells, due to the fact that brittleness of ovule tissues is increased under the low-temperature condition, under the condition of mechanical oscillation together with glass beads, besides shedding of fiber cells, other non-fiber tissues are also shed in a large amount, and the specificity of an obtained RNA sample is greatly reduced.
The difficulty in solving the above problems and defects is: before and after cotton blossoming, only about 30% of epidermal cells of ovule epidermis are differentiated and protuberant to form fiber cells, and at this stage, the fiber cells, the ovule epidermal cells and the ovule epididymis tissues are combined relatively tightly, so that a large amount of specific cotton fiber cells are difficult to separate.
The significance of solving the problems and the defects is as follows: cotton fiber cells are one of the longest single cells in the world and are one of the important materials for studying cells. The differentiation and initiation of the fiber cells are crucial to the fiber development, but the research on the differentiation and initiation of the fiber cells is not clear up to now, the separation of the early specific cotton fiber cells and the acquisition of high-quality early transcriptional group data of the cotton fiber cell development contribute to the disclosure of the fiber differentiation and initiation processes, and simultaneously contribute to the excavation of fiber initiation related genes, thereby laying a foundation for the cultivation of high-quality cotton fiber varieties.
Disclosure of Invention
Aiming at the problems in the prior art, the application provides a method for extracting RNA in the early development stage of cotton fiber cells.
The application is realized by that, a method for extracting RNA in early development stage of cotton fiber cells comprises the following steps:
step one, obtaining early complete ovules. The cotton was marked for the growing period (ovary one day before flowering was-1 DPA, ovary one day after flowering was 0DPA, ovary one day after flowering was 1DPA, ovary two days after flowering was 2DPA, ovary three days after flowering was 3DPA, ovary four days after flowering was 4DPA, and ovary five days after flowering was 5 DPA). Several ovaries of different developmental stages were taken, the walls of the ovaries were carefully dissected in an ultraclean bench with RNase-Free forceps and razor blades, the handles were gently grasped with forceps, and the intact ovules were removed.
And step two, vacuumizing and cracking cells in the early development stage of the cotton fibers.
Placing the ovule into a sterile centrifuge tube filled with lysis solution, opening the centrifuge tube, placing into a dryer, vacuumizing to-0.9 Mpa for 10min, and gently shaking for 5 min. Placing into 4 deg.C centrifuge at 12000rpm for 5min, and collecting supernatant.
Lysis solution: 1/4 volume TE [ TE buffer (pH 8.0): 10mM Tris (pH8.0), 1mM EDTA (pH8.0) ], 3/4 vol Binding Buffer [ 66% guanidine hydrochloride, 2% Triton X-100, 5% beta-mercaptoethanol ].
And step three, extracting early-stage RNA of fiber development by an adsorption elution method.
Transferring the supernatant to a filter column CS (with the aperture of 20-25um) for filtering, adding 0.4-time volume of absolute ethyl alcohol into the filtered supernatant for precipitating nucleic acid, transferring the obtained solution into an adsorption column CR3 (with the aperture of 10-15um) for collection, adding a deproteinizing solution RW1 to the center of the adsorption column CR3 for washing protein, adding DNase I working solution to the center of the adsorption column for digesting DNA, adding a deproteinizing solution RW1 and a rinsing solution RW for washing other impurities except the nucleic acid, and finally collecting the eluted RNA solution.
And step four, detecting the concentration and purity of the RNA. The purity and concentration of the 1. mu.L RNA solution were checked by Nanodrop 2000.
The invention also aims to provide a device for extracting RNA in early development stage of cotton fiber cells, which is used for implementing the method for extracting RNA in early development stage of cotton fiber cells.
The variety used by the method is upland cotton TM-1, and the method is suitable for any cotton variety. Can quickly extract cotton fiber cell development early specific RNA which is not easy to separate in the periods of-1 DPA, 0DPA, 1DPA, 2DPA, 3DPA, 4DPA and 5DPA, and meets the requirement of a transcription and tissue library construction.
By combining all the technical schemes and the embodiments, the application has the advantages and positive effects that:
(1) the method can extract the high-quality RNA in the early development stage of cotton fiber cells, and is the most accurate method for extracting the RNA in the early development stage of cotton fibers.
(2) Comparison analysis of transcriptome data obtained by the application and published transcriptome data shows that the fiber-related gene specificity expression in the transcriptome data is higher, which indicates that the transcriptome data obtained by the application is better.
(3) The application firstly proposes that the RNA in the early development stage of cotton fiber cells is extracted by using a vacuum cracking method, the extracted RNA has high quality, and the transcriptome data is more accurate.
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In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
FIG. 1 is a flowchart of a method for extracting RNA in early developmental stage of cotton fiber cells according to an embodiment of the present invention.
FIG. 2 is a graph of the results of environmental scanning imaging of 0DPA cotton ovule surfaces prior to treatment with lysis buffer according to an embodiment of the present invention.
Fig. 3 is a graph of the results of environmental scanning imaging of the surface of 0DPA cotton ovule after treatment with lysis buffer according to an embodiment of the present invention.
FIG. 4 is a semi-thin section of an unfrozen ovule according to an embodiment of the present invention.
FIG. 5 is a semi-thin slice of an ovule subjected to vacuum cracking according to an embodiment of the present invention.
FIG. 6 is a comparison graph of the expression level of specific genes in early stage of cotton Fiber development provided in the example of the present invention (Fiber represents the gene expression level data of cells in early stage of cotton Fiber development obtained in the present invention, and Ovule represents the publicly published gene expression level data of whole ovules).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a method for extracting RNA in early development stage of cotton fiber cells, and the invention is described in detail with reference to the attached drawings.
As shown in FIG. 1, the present invention provides a method for extracting RNA in early developmental stage of cotton fiber cell, comprising:
s101, obtaining early intact ovules. The cotton was marked for the growing period (ovary one day before flowering was-1 DPA, ovary one day after flowering was 0DPA, ovary one day after flowering was 1DPA, ovary two days after flowering was 2DPA, ovary three days after flowering was 3DPA, ovary four days after flowering was 4DPA, and ovary five days after flowering was 5 DPA). Several ovaries of different developmental stages were taken, the walls of the ovaries were carefully dissected in an ultraclean bench with RNase-Free forceps and razor blades, the handles were gently grasped with forceps, and the intact ovules were removed.
S102, vacuumizing and cracking cells in the early development stage of cotton fibers. Placing the ovule into a sterile centrifuge tube filled with lysis solution, opening the centrifuge tube, placing into a dryer, vacuumizing to-0.9 Mpa for 10min, and gently shaking for 5 min. Placing into 4 deg.C centrifuge at 12000rpm for 5min, and collecting supernatant. Lysis solution: 1/4 volume TE [ TE buffer (pH 8.0): 10mM Tris (pH8.0), 1mM EDTA (pH8.0) ], 3/4 vol Binding Buffer [ 66% guanidine hydrochloride, 2% Triton X-100, 5% beta-mercaptoethanol ].
S103, extracting early-stage RNA of fiber development by an adsorption elution method. Transferring the supernatant to a filter column CS (with the aperture of 20-25um) for filtering, adding 0.4-time volume of absolute ethyl alcohol into the filtered supernatant for precipitating nucleic acid, transferring the obtained solution into an adsorption column CR3 (with the aperture of 10-15um) for collection, adding a deproteinizing solution RW1 to the center of the adsorption column CR3 for washing protein, adding DNase I working solution to the center of the adsorption column for digesting DNA, adding a deproteinizing solution RW1 and a rinsing solution RW for washing other impurities except the nucleic acid, and finally collecting the eluted RNA solution.
And S104, detecting the RNA concentration and purity. The purity and concentration of the 1. mu.L RNA solution were checked by Nanodrop 2000.
Those of ordinary skill in the art of the methods provided herein may also perform other steps, and the invention of fig. 1 is provided as a specific example only.
The invention is further described with reference to specific examples.
Example 1: extraction and quality detection of fiber RNA of cotton variety TM-1 (from research institute of Cotton, national academy of agricultural sciences) at different development stages-1 DPA to 5 DPA.
1) The materials are obtained. 40 cotton ovaries of-1 DPA, 0DPA, 1DPA, 2DPA, 3DPA, 4DPA and 5DPA were respectively selected from TM-1 plants.
2) And (4) cracking. And (4) taking 7 centrifuge tubes with the volume of 15mL, adding 8mL of prepared lysate into each centrifuge tube, and marking. Carefully cutting open the ovary wall of the flower in the step 1 by using a blade on a clean bench, gently clamping a handle of the bead by using a pair of tweezers, taking out the complete ovule and putting the complete ovule into a corresponding centrifuge tube.
3) And (6) vacuumizing. And (3) opening the cover of the centrifugal tube in the step (2), putting the centrifugal tube into a dryer, vacuumizing to-0.9 Mpa for 10-20 min (-1DPA sample is vacuumized for 10min, 0DPA sample is vacuumized for 12min, 1DPA sample is vacuumized for 14min, 2DPA sample is vacuumized for 16min, 3DPA sample is vacuumized for 18min, 4DPA sample and 5DPA sample are vacuumized for 20min), taking out, and gently shaking for 5 min. Placing into 4 deg.C centrifuge at 12000rpm for 5min, and collecting supernatant.
4) And collecting the supernatant. The supernatant was transferred to filtration column CS and centrifuged at 12000rpm for 2min, and the sample supernatant was filtered several times. Carefully aspirate the pool into a new 15mL RNase-Free centrifuge tube.
5) Slowly adding 0.4 times volume of anhydrous ethanol, mixing, transferring the obtained solution into adsorption column CR3 for multiple times, centrifuging at 12000rpm for 1min, and removing waste liquid in the collection tube.
6) 350 μ L deproteinizing solution RW1 was added to the center of adsorption column CR3, centrifuged at 12000rpm for 1min, the waste liquid in the collection tube was discarded, and adsorption column CR3 was returned to the collection tube.
7) 60 μ L of DNase I stock solution was put into a new 1.5mL RNase-Free centrifuge tube, 420 μ L of RDD buffer was added, and gently mixed.
8) Add 80. mu.L of each DNase I (0.34U/ul) to the center of the column and leave it at room temperature for 15 min.
9) 350 μ L deproteinizing solution RW1 was added to the center of adsorption column CR3, centrifuged at 12000rpm for 1min, the waste liquid in the collection tube was discarded, and adsorption column CR3 was returned to the collection tube.
10) Adding 500 μ L of rinsing solution RW into adsorption column CR3, centrifuging at 12000rpm for 1min, removing waste liquid from the collection tube, and returning adsorption column CR3 to the collection tube.
12) Repeating the previous step.
12) Centrifuging at 12000rpm for 2min, placing adsorption column CR3 into a new 1.5mL RNase-Free centrifuge tube, and adding 30 μ L RNase-Free ddH dropwise into the middle part of the adsorption membrane2O, standing at room temperature for 2min, and centrifuging at 12000rpm for 1min to obtain an RNA solution.
13) And (5) quality detection. Purity (260/280, 260/230) and concentration were measured using Nanodrop2000, and total and RIN values were accurately measured using Agilent 2100RNA 6000Nano kit, and samples were graded according to purity, concentration, total and RIN values.
Example 1 the improvement of vacuuming treatment is made in the cracking part, and the extracted RNA in the early development stage of cotton fiber is more accurate and specific. The quality inspection results show that 7 RNA samples are primary quality samples (Table 1), which shows that the quality of the RNA extracted in the early stage of fiber development is high, and the requirement of transcriptome library construction can be met.
TABLE 1 RNA quality test results of early cotton fiber development
Example 2: observation of ovule tissue imaging by scanning electron microscope environment
Taking 1 TM-10 DPA cotton ovary, carefully dissecting the ovary wall with a blade, gently clamping the bead stalk with a forceps, taking out 6 complete ovules with the same development state, and directly scanning in an electron microscope environment without treatment for 3 ovules. The other 3 are subjected to cracking treatment, the obtained ovule is put into a 1.5mL centrifuge tube containing 800 μ L of cracking solution, vacuum pumping is carried out until the pressure is-0.9 Mpa and the pressure is 12min, vortex oscillation is carried out for 5min, and ddH2And cleaning for three times, scanning an electron microscope environment, and multiplying power by 1000 times.
The results of electron microscopy showed that the blastomere epidermal cell process without lysis treatment was normal and the fibroblasts were uniformly and fully distributed in the epidermis (FIG. 2). The shrinkage of the vacuum-lysed ovule epidermal tissue was evident as the ovule epidermal cells were lysed (FIG. 3).
Example 3: observation of semi-thin section of ovule
1 of TM-10 DPA cotton ovaries was taken, the ovary wall was carefully dissected with a blade, the petioles were gently grasped with forceps, 6 whole ovules in the same developmental state were removed, and 3 specimens were directly fixed without treatment. And performing cracking treatment on the other 3 ovules, putting the ovules into a 1.5mL centrifuge tube containing 800 mu L of cracking solution, vacuumizing to-0.9 Mpa for 12min, performing vortex oscillation for 5min, fixing the sample by using a fixing solution, slicing and observing.
The semi-thin slicing procedure was as follows:
1) and (5) fixing the sample. The ovule is placed into a 2mL centrifuge tube containing 1mL fixative (the fixative components are 4% paraformaldehyde and 5% glutaraldehyde), and the tube is evacuated to-0.9 MPa until the ovule is completely immersed in the fixative.
2) And (5) cleaning. 0.1M Pbs (phosphate buffered saline) was washed 3 times for 15min each.
3) And (4) dehydrating. Sequentially dehydrating with 30% ethanol, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, 100% ethanol, and 100% ethanol for 15min each time.
4) And (4) soaking. Anhydrous ethanol: LR white (1: 1) was soaked for 1h, first for 3-5h, and second overnight.
5) And (4) embedding and polymerizing. The sample is put into a capsule, filled with LR white and put into a polymerization box at 70 ℃ for 24 h.
6) And (6) slicing. And cutting the embedded sample in the step by an ultrathin microtome, wherein the section thickness is 500-1000 nm.
7) And (6) dyeing. 0.05% crystal violet stains the good slices.
8) And (6) observing. The image was photographed under an optical microscope at a magnification of 40.
The results show that the lysate successfully digested ovule epidermal cells, i.e., cells in the early stages of cotton fiber development. In the ovule treated by vacuum lysis, the outermost cells were significantly exfoliated and broken, while the inner cells were not damaged at all (FIG. 5), and the unlysed sample was normal (FIG. 4).
Example 4: transcriptome data-specific comparative analysis
Transcriptome sequencing is carried out on the obtained cotton fiber early cell RNA in different developmental stages, and specific genes GhMYB25(GH _ D04G2108), GhMYB25-like (GH _ D12G1824), GhJAZ2(GH _ D06G0835), GhRDL1(GH _ A05G0504) and GhDET2(GH _ A04G1074) which are reported to participate in the cotton fiber cell initiation process are selected as objects, and the expression quantity in the whole ovule (published) and cotton fiber early cell specific (the invention) transcriptome is contrastively analyzed. The results show that the expression levels of 6 genes in the transcriptome of the invention at various time points are higher than the corresponding expression levels in the whole-ovule transcriptome, and high specificity is presented. Such as: GhMYB25-like (-1DPA), GhDET2(3DPA and 5DPA), GhJAZ2(-1DPA, 0DPA, and 1DPA), GhMYB25(-1DPA, 0DPA, 1DPA, 3DPA, and 5DPA), and GhRDL1(-1DPA, 0DPA, 1DPA, 3DPA, and 5DPA) (FIG. 6).
In the description of the present invention, "a plurality" means two or more unless otherwise specified; the terms "upper", "lower", "left", "right", "inner", "outer", "front", "rear", "head", "tail", and the like, indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are only for convenience in describing and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed in a particular orientation, and be operated, and thus, should not be construed as limiting the invention. Furthermore, the terms "first," "second," "third," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (2)
1. A method for extracting RNA in early development stage of cotton fiber cells, which comprises the following steps:
step one, obtaining early intact ovules: taking a plurality of ovaries in different development stages, and taking complete ovules;
step two, vacuumizing and cracking cells at the early development stage of cotton fibers: putting the ovule into lysis solution, vacuumizing, centrifuging, and taking supernatant;
step three, extracting early RNA of fiber development by an adsorption elution method: filtering the supernatant, adding absolute ethyl alcohol into the filtered supernatant to precipitate nucleic acid, collecting the obtained solution, adding a deproteinizing solution RW1 to wash protein, adding DNase I working solution to digest DNA, adding a deproteinizing solution RW1 and a rinsing solution RW to wash other impurities except the nucleic acid, and finally collecting the eluted RNA solution;
step four, detecting the concentration and purity of RNA;
in the first step, the cotton is marked in the growing period, the ovary of the day before flowering is-1 DPA, the ovary of the day after flowering is 0DPA, the ovary of the day after flowering is 1DPA, the ovary of the two days after flowering is 2DPA, the ovary of the three days after flowering is 3DPA, the ovary of the four days after flowering is 4DPA, and the ovary of the five days after flowering is 5 DPA;
in the second step, putting the ovules into a sterile centrifuge tube filled with a lysis solution, opening a cover of the centrifuge tube, putting the centrifuge tube into a dryer, vacuumizing to-0.9 Mpa, vacuumizing a-1 DPA sample for 10min, vacuumizing a 0DPA sample for 12min, vacuumizing a 1DPA sample for 14min, vacuumizing a 2DPA sample for 16min, vacuumizing a 3DPA sample for 18min, vacuumizing a 4DPA sample and a 5DPA sample for 20min, and gently oscillating for 5 min; placing into 4 deg.C centrifuge at 12000rpm for 5min, and collecting supernatant;
in the second step, the lysis solution consists of 1/4 volumes of TE solution and 3/4 volumes of Binding Buffer solution;
the TE solution consisted of 10mM Tris, pH8.0, and 1mM EDTA;
the Binding Buffer solution consists of 66% guanidine hydrochloride, 2% Triton X-100 and 5% beta-mercaptoethanol;
in the third step, adding 0.4 volume times of absolute ethyl alcohol into the filtered supernatant to precipitate nucleic acid, and transferring the obtained solution into an adsorption column CR3 for collection;
in the fourth step, 1 mu of LRNA solution is taken and the concentration and the purity are detected by using Nanodrop 2000.
2. The method for extracting early developmental RNA from cotton fiber cells of claim 1, wherein the method for extracting early developmental RNA from cotton fiber cells further comprises:
1) collecting materials, and respectively collecting 40 cotton ovaries of-1 DPA, 0DPA, 1DPA, 2DPA, 3DPA, 4DPA and 5DPA from a TM-1 plant;
2) cracking, namely taking 7 centrifuge tubes with the volume of 15mL, respectively adding 8mL of prepared lysate, and marking; taking out the complete ovules of the flowers in the step 1 from a super clean workbench, and putting the complete ovules into corresponding centrifuge tubes;
3) vacuumizing, namely, opening the cover of the centrifuge tube in the step 2, putting the centrifuge tube into a dryer, vacuumizing to-0.9 Mpa, vacuumizing for 10min for a-1 DPA sample, vacuumizing for 12min for a 0DPA sample, vacuumizing for 14min for a 1DPA sample, vacuumizing for 16min for a 2DPA sample, vacuumizing for 18min for a 3DPA sample, vacuumizing for 20min for 4DPA and 5DPA samples, taking out, and gently oscillating for 5 min; placing into 4 deg.C centrifuge at 12000rpm for 5min, and collecting supernatant;
4) collecting supernatant, transferring the supernatant to a filter column CS, centrifuging at 12000rpm for 2min, and filtering the sample supernatant for multiple times; sucking the collected liquid into a new 15mL RNase-Free centrifuge tube;
5) slowly adding 0.4 times volume of anhydrous ethanol, mixing, transferring the obtained solution into adsorption column CR3 for multiple times, centrifuging at 12000rpm for 1min, and pouring off waste liquid in the collection tube;
6) adding 350 μ L deproteinized solution RW1 into the center of adsorption column CR3, centrifuging at 12000rpm for 1min, pouring off waste liquid in the collection tube, and placing adsorption column CR3 back into the collection tube;
7) putting 70 mu L of DNase I stock solution into a new 1.5mL RNase-Free centrifuge tube, adding 490 mu L of RDD buffer solution, and uniformly mixing;
8) adding 80 μ L of DNase I into the center of the adsorption column respectively, and standing at room temperature for 15 min;
9) adding 350 μ L deproteinized solution RW1 into the center of adsorption column CR3, centrifuging at 12000rpm for 1min, pouring off waste liquid in the collection tube, and placing adsorption column CR3 back into the collection tube;
10) adding 500 μ L of rinsing solution RW into adsorption column CR3, centrifuging at 12000rpm for 1min, pouring off waste liquid in the collection tube, and placing adsorption column CR3 back into the collection tube;
11) repeating the previous step;
12) centrifuging at 12000rpm for 2min, placing adsorption column CR3 into a new 1.5mL RNase-Free centrifuge tube, and adding 30 μ L RNase-Free ddH dropwise into the middle part of the adsorption membrane2O, standing at room temperature for 2min, and centrifuging at 12000rpm for 1min to obtain an RNA solution;
13) quality test, purity and concentration test by using Nanodrop 2000.
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