CN119464274B - Method for separating, extracting and purifying nucleic acid from different cell components - Google Patents
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Abstract
The invention belongs to the technical field of nucleic acid extraction, and particularly relates to a method for separating, extracting and purifying nucleic acid from different cell components. The invention develops a separation and purification method suitable for liquid-phase RNA such as cytoplasmic component, nuclear free component and the like, enables the extracted RNA to be compatible with the highest standard of nucleic acid mass spectrum and high-flux sequencing, has simpler operation flow, shorter time, lower cost and better and more efficient extraction effect, and simultaneously develops a corresponding nuclear DNA/RNA separation buffer and a high-efficiency nucleic acid purification flow on the basis, so that the genomic DNA can be completely extracted with high quality and high purity while high-quality chromatin RNA is obtained, and information of another layer, namely genomic DNA information, is provided for a classical component separation scheme of RNA dynamic biology, thereby enabling all nucleic acid information of each component of cells to be separated and purified with high efficiency.
Description
Technical Field
The invention belongs to the technical field of nucleic acid extraction, and particularly relates to a method for separating, extracting and purifying nucleic acid from different cell components.
Background
The separation of the cellular components of nucleic acids, in particular RNA, is of great importance for studying the function of RNA. In eukaryotic cells, RNA synthesis, processing and function take place in specific subcellular compartments. By fractionation of the biochemical components of the cells, the different stages of the RNA life cycle can be analyzed in depth. At present, the cell component separation technology is widely applied to the research on the synthesis, processing and transportation of biomolecules and organelles, and in recent years, the technology is combined with a novel deep sequencing technology, so that unprecedented deep insight is provided for the generation and the functions of primary RNA transcripts.
The basic principle and operation of RNA cell component separation is that firstly, cells are lysed by mild detergent, cell organelles and other cell components are released into suspension while maintaining the integrity of nuclear membrane, and primary purified cell nucleus is obtained by 24% sucrose centrifugation and precipitation, and the supernatant is further centrifuged at high speed to obtain cytoplasmic extract. The second step of component separation was performed using the method developed in 1994 by Wuarin and Schibler, which aimed at separating chromatin and nucleoplasm complexed with nascent RNA, to investigate whether RNA splicing co-occurs with transcription. The method specifically comprises the steps of lightly suspending settled cell nuclei in buffer solution containing 50% of glycerol, and rapidly adding 1% of Igepal 630 and 1M of urea to realize the lysis, wherein the key innovation is to use urea to replace ionic detergents to break nuclear membranes, so that the combination of histones including histone H1 and genomic DNA can be maintained, a compact chromatin structure is reserved, and a chromatin-RNA complex can be precipitated by a table centrifuge. Finally, RNA can be recovered from each of the isolated fractions by phenol-chloroform extraction or TRIZOL extraction. Currently, this approach has been optimized by a number of laboratories and has shown significant advantages in studying neonatal transcription, exon usage, splicing kinetics, RNA processing, transport and degradation, especially in capturing short-lived chromatin-associated primary transcripts (e.g., microRNA precursors). The technology has important value and wide applicability in the research of RNA dynamic biological process because of simple operation and suitability for various cell types.
Currently, in terms of RNA extraction after component separation, cytoplasmic component RNA is present in the cytoplasmic lysate, nuclear free RNA component is also present in the lysate in the liquid phase, whereas chromatin RNA (precipitated phase) can be directly solubilized with TRIZOL and purified and separated using classical TRIZOL RNA extraction methods. However, for RNA (cytoplasmic fraction and nuclear free fraction RNA) present in the liquid phase lysate, since it is unstable and easily degraded, it is necessary to purchase TRIZOL-LS reagent for extracting the liquid phase, and 0.25mL of liquid phase solution is required to use 0.75mL of TRIZOL-LS reagent, on the one hand, TRIZOL-LS is expensive, and on the other hand, TRIZOL-LS is huge in volume after treatment, which is unfavorable for the separation and purification of the subsequent RNA. Meanwhile, the RNA cell fraction separation method often results in loss of genomic DNA information of the cell tissue due to the use of a specific chromatin structure-maintaining reagent (intended to separate the RNA that actually binds to chromatin) and the use of TRIZOL extraction. Therefore, there is a need to develop a new method for isolating RNA cellular components, which can simultaneously extract genomic DNA from the precipitate of isolated chromatin RNA, and which can provide not only transcriptome information of each cellular component but also genomic information corresponding thereto.
In our previously disclosed patent CN118240814a, a principle and method of distinguishing DNA from RNA in the liquid phase in vitro was established, but this method is only applicable in the liquid phase in vitro and has not been applied in real cell samples, and thus it is doubtful whether it is equally possible to distinguish DNA from RNA in real cell samples and in complex treated chromosomal cell pellets. Moreover, one of the most important problems is that DNA and RNA in complex cell samples or cell pellets often do not exist in the aqueous phase at the same time, and thus are not suitable for practical cell samples in principle. For example, after the chromatin precipitate phase separated from the traditional classical cellular components is treated by a specific extraction reagent for compacting chromatin structure, chromatin RNA and DNA exist in a compact cellular precipitate, wherein RNA can be re-dissolved in a liquid phase through TRIZOL, but DNA in the cellular precipitate phase cannot be re-dissolved in the liquid phase after the use of the TRIZOL reagent, so that high-quality and high-purity DNA cannot be prepared. Therefore, there is an urgent need to develop a DNA/RNA extraction reagent that allows both RNA and DNA in the cell pellet phase to be efficiently reconstituted in the liquid phase, thereby facilitating the subsequent silica gel purification procedure.
In conclusion, for RNA components existing in the liquid phase lysate, the traditional separation and purification flow is more than 1h, meanwhile, the separation reagent is expensive, and the volume is huge after the separation reagent is added, so that the experimental operation is complicated. RNA in the liquid phase lysate is also easily degraded, so that development of a method compatible and suitable for rapid separation and purification of RNA in the liquid phase lysate is highly demanded. Meanwhile, in the traditional classical RNA cell component separation scheme, the problem of losing genome DNA information is not solved so far, and if the corresponding genome DNA can be simultaneously extracted and separated in the RNA cell component separation scheme, nucleic acid (RNA and DNA) components in each cell component can be completely separated and purified basically, so that the complete genome information and transcriptome information can be obtained. In addition, although a method for efficiently separating and purifying DNA and RNA is established in our previous patent CN118240814A, it is only directed to DNA and RNA existing in the in vitro liquid phase, and has no ability to separate and purify RNA and DNA in the chromatin precipitation phase. Therefore, it is necessary to develop a method for separating and purifying RNA which is suitable for cytoplasmic components, nuclear free components, chromatin and other liquid phase or precipitate phase simultaneously, and has the advantages of simpler operation flow, shorter time, lower cost, better extraction effect and higher efficiency.
Disclosure of Invention
In order to solve the problems of complex, time-consuming, high-cost and easy degradation of RNA in the classical RNA cell component separation scheme, the invention develops a separation and purification method suitable for the liquid-phase RNA such as cytoplasmic component and nuclear free component, wherein the recovery rate of the cytoplasmic RNA is 72.3-87.8%, the recovery rate of the nuclear free RNA is 75.9-97.6%, the purity is high, the OD 260/280 (more than 1.8) and the OD 260/230 (more than 1.8) parameters are good, and the method can be compatible with the highest standards of nucleic acid mass spectrum and high-flux sequencing. Meanwhile, the purification time of cytoplasmic RNA and nuclear free RNA is only 4min, the purification time of chromatin RNA is 16min, the operation is simple and convenient, the duration is short, and the method can be suitable for the preparation requirement of high-flux samples. In addition, for DNA in chromatin precipitation components, a corresponding nucleoplasm DNA/RNA separation buffer is also developed, and can separate and extract chromatin RNA and genome DNA efficiently at the same time, and the purification and separation time of the genome DNA is 14min. Therefore, the scheme of the invention can effectively separate and purify all nucleic acid components in each component of the cells, and provides technical support for the research field of RNA dynamic biology.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides a kit for separating, extracting and purifying nucleic acid from different cell components, wherein the kit comprises an RNA binding solution, absolute ethyl alcohol, an RNA rinsing solution 1 solution, an RNA rinsing solution 2 solution, a nuclear DNA/RNA separation buffer, chloroform and a DNA rinsing solution;
The RNA binding solution consists of 1-8M guanidine thiocyanate and 1M Tris-HCl, wherein the pH=7.4, the RNA rinsing solution 1 consists of ethanol and guanidine hydrochloride, the RNA rinsing solution 2 consists of ethanol solution, the nucleoplasm DNA/RNA separation buffer consists of a lysate and a TRIZOL reagent, the lysate consists of 4M guanidine thiocyanate and 1M Tris-HCl, the pH=7.4, and the DNA rinsing solution consists of ethanol and NaCl.
Preferably, the RNA rinse 1 solution is composed of 80% ethanol and 2M guanidine hydrochloride, the RNA rinse 2 solution is 80% ethanol, the nucleoplasm DNA/RNA separation buffer is composed of a lysate and TRIZOL reagent according to a volume ratio of 1:2, and the DNA rinse is composed of 80% ethanol and 100mM NaCl.
In a second aspect, the present invention provides a method for separating, extracting and purifying nucleic acid from different cellular components, specifically, separating, extracting and purifying nucleic acid from different cellular components using the kit according to the first aspect, comprising the steps of:
S1, separating cytoplasmic components, nuclear free components and chromosome components from cells;
S2, extracting RNA of cytoplasmic components or nuclear free components:
S21, adding RNA binding solution into cytoplasmic components or nuclear free components, and then adding absolute ethyl alcohol for uniform mixing;
s22, incubating the mixed solution of the S21 with a silica gel column to combine RNA with the silica gel column;
S23, adding the RNA rinsing liquid 1 solution into the silica gel column of the S22 for rinsing;
s24, adding the solution of the RNA rinsing liquid 2 into the silica gel column of S23 for rinsing, and then performing air-throwing and pure water elution to obtain an RNA sample.
S3, co-extraction of chromatin-associated RNA and genomic DNA:
s31, separating buffer by using nucleoplasm DNA/RNA to dissolve chromosome components, adding chloroform, centrifuging, mixing uniformly and layering;
s32, taking supernatant in the step S31, and incubating with a silica gel column to combine DNA with the silica gel column;
S33, adding a DNA rinsing liquid into the silica gel column of S32 for rinsing, and then performing air-throwing and pure water elution to obtain a genome DNA sample;
S33, collecting the flow-through liquid of S32, adding absolute ethyl alcohol, incubating the flow-through liquid with a new silica gel column to combine RNA with the silica gel column, and then processing according to S23 and S24 to obtain a chromatin RNA sample.
The invention develops a liquid-phase RNA separation and purification method suitable for cytoplasmic components, nuclear free components and the like, so that the operation flow is simpler, the time is shorter, the cost is lower, and the extraction effect is better and more efficient. Meanwhile, a nucleoplasm DNA/RNA separation buffer is developed, and a high-efficiency nucleic acid purification process is matched, so that the high-quality chromatin RNA is obtained, meanwhile, the complete high-quality high-purity genomic DNA can be extracted, and the other layer of information, namely genomic DNA information, is provided for a classical component separation scheme of RNA dynamic biology, so that the high-efficiency separation and purification of all nucleic acid information of all components of a cell can be realized.
Preferably, in S21, the RNA binding solution added to the cytoplasmic fraction consists of 5M guanidine thiocyanate and 1M Tris-HCl, ph=7.4, and the RNA binding solution added to the nuclear free fraction consists of 6-7M guanidine thiocyanate and 1M Tris-HCl, ph=7.4.
Preferably, in S21, the volume ratio of cytoplasmic fraction or nuclear free fraction, RNA binding solution, and absolute ethanol is 1:1:2-4.
Preferably, in S31, the volume ratio of the nucleoplasmic DNA/RNA isolation buffer to chloroform is 4-6:1.
Preferably, in S33, the volume ratio of the flow-through liquid to the absolute ethanol is 1:1-2.
Preferably, in S33, 10000-20000g of rinsing liquid is rinsed for 30S-1min, 1-2min is air-thrown, 10000-20000g of pure water is eluted for 1-3min after the DNA rinsing liquid is added.
Preferably, in S24, 10000-20000g of rinsing liquid 2 solution is added, and then rinsing is performed for 30S-1min, 1-2min is performed for air-throwing, and 10000-20000g of pure water is eluted for 1-3min.
Preferably, in S22, S32, S33, after incubation with silica gel column, 10000-20000g is centrifuged for 30S-1min to bind RNA/DNA to silica gel column.
Preferably, in S23, 10000-20000g is rinsed for 30S-1min after adding the RNA rinsing liquid 1 solution.
Preferably, the separation of the cytoplasmic, nuclear free and chromosomal components from the cells of S1 comprises in particular the following steps:
S11, washing cells with ice-cold PBS/1 mM EDTA buffer solution, and centrifuging to collect cell sediment;
S12, adding ice-cold lysis buffer 1 into the cell sediment of S11, incubating on ice, slightly pipetting, uniformly mixing the lysis solution, covering the lysis solution on an ice-cold sucrose cushion, centrifuging, collecting the supernatant as a cytoplasmic component for later use, and allowing the rest cell nucleus sediment to enter the next step, wherein the lysis buffer 1 consists of 10mM Tris-HCl,0.05% IGEPAL CA-630 and 150 mM NaCl, 2 mu L of RNase inhibitor is added per 1mL, the pH=7.5, and the sucrose cushion is prepared by dissolving the nuclease-free sucrose in the lysis buffer 1 according to the mass percentage of 20-30%;
S13, adding ice-cold PBS/1 mM EDTA buffer solution into the cell nucleus precipitate obtained in S12, removing PBS/EDTA after not disturbing the precipitate, re-suspending the cell nucleus precipitate in precooled glycerol buffer solution, adding an equal volume of ice-cold nucleus lysis buffer solution after uniformly mixing, incubating, centrifuging to collect supernatant as a free component of the nucleus for later use, flushing the precipitate with the ice-cold PBS/1 mM EDTA buffer solution, and collecting the precipitate as a related component of the chromosome, wherein the glycerol buffer solution consists of 20 mM Tris-HCl,75 mM NaCl,0.5 mM EDTA,0.85 mM DTT,0.125 mM PMSF,50% glycerol, the pH=7.9, and the nucleus lysis buffer solution consists of 10mM HEPES,1 mM DTT,7.5 mM MgCl 2, 0.2 mM EDTA,0.3M NaCl,1M urea and 1% IGEPAL CA-630, and the pH=7.6.
More preferably, in S11, 1mL ice-cold PBS/1 mM EDTA buffer is added to every 5X 10 6-1×107 cells.
Compared with the prior art, the invention has the beneficial effects that:
The invention develops a separation and purification method suitable for liquid-phase RNA such as cytoplasmic components, nuclear free components and the like, so that the extracted RNA can be compatible with the highest standard of nucleic acid mass spectrum and high-throughput sequencing, the operation flow is simpler, the time is shorter, the cost is lower, the extraction effect is better and more efficient, and meanwhile, a corresponding nuclear DNA/RNA separation buffer is also developed on the basis, and can simultaneously separate and extract chromatin RNA and genome DNA efficiently, so that the whole nucleic acid information of each component of the cell can be separated and purified efficiently.
The invention develops a method compatible with the subsequent efficient purification of RNA in the classical RNA cell component separation experiment, provides a scheme flow for efficiently purifying cytoplasmic RNA components and a method for efficiently purifying nuclear free RNA components, has the advantages of about 4min of whole RNA extraction and purification time, high RNA purity and suitability for the highest standard of high-throughput sequencing. Meanwhile, a nucleoplasm DNA/RNA separation buffer is developed, DNA and RNA components are extracted from chromatin precipitation simultaneously, the integrity purity of the DNA and RNA reaches the highest standard, and the subsequent high-throughput sequencing requirement can be met. The method is characterized in that the attempt of separating DNA from chromatin precipitation is initiated, the purification time of the whole DNA and RNA is 14min for DNA and 16min for RNA, the classical scheme of RNA component separation is greatly perfected, and technical support is provided for the development of the RNA dynamic biology field.
Drawings
FIG. 1 shows the effect of different RNA binding solutions on the extraction of RNA from cytoplasmic fractions (lanes 1-8 show experimental treatments of different RNA binding solutions, see in particular Table 1, M is Marker);
FIG. 2 shows the effect of different RNA binding solutions on the extraction of RNA from nuclear free fractions (lanes 1-8 show experimental treatments of different RNA binding solutions, see in particular Table 2, M is Marker);
FIG. 3 shows the effect of nucleic acid extraction of different cellular components (lanes 1-4 show RNA/DNA of different cellular components, see in particular Table 3, M is Marker).
In FIGS. 1 to 3, M is Marker, and is 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp in order from large to small.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
The purification column used in the invention is derived from Zymo research, the model is C1004-50, and any silica gel column with guaranteed quality in the market meets the requirements.
Aiming at the problem that the liquid-phase RNA extraction and purification are difficult after the RNA cell component is fractionated, the invention develops a silica gel flow scheme for efficiently compatible liquid-phase RNA extraction, and can provide reference for the establishment of other efficient nucleic acid fractionation schemes in liquid-phase lysate.
In the classical RNA cell component separation scheme, cytoplasmic components and nuclear free components exist in liquid phase lysate, and are usually treated by TRIZOL-LS reagent, so that on one hand, TRIZOL-LS reagent is expensive, on the other hand, 3 times of TRIZOL-LS reagent is required to be added into one-time volume of liquid phase lysate, and the volume of the solution is huge, so that the subsequent extraction and purification are very complicated. Therefore, the invention is based on the principle of RNA silica gel extraction (RNA can be combined with a silica gel column and separated under the action of 1-2 times of absolute ethyl alcohol in a certain salt solution), and an efficient nucleic acid separation and purification scheme is excavated from the compatibility problem of separating a lysate from RNA cell components and a silica gel column system, and firstly, corresponding RNA binding solution is developed, meanwhile, components of cytoplasmic lysate and nuclear free lysate are considered to be very complex, the subsequent silica gel purification is facilitated, the reagent cost is reduced, the formula of the RNA binding solution is further simplified, and finally, the compatibility of the cytoplasmic lysate and the nuclear free lysate with the silica gel column can be realized only by using the single salt component of guanidine thiocyanate. As can be seen from examples 1 and 2, the recovery rate of 72.3-87.8% of RNA in the cytoplasmic lysate can be realized by using simple RNA binding solution, namely 1-8M guanidine thiocyanate, 1M Tris-HCl and PH=7.4, the recovery rate of the nuclear free lysate is more high up to 75.9% -97.6%, the purity of the separated RNA is high, OD 260/280 is more than 1.8, and OD 260/230 is more than 1.8, and the purity requirement of high-throughput sequencing can be met.
For the precipitated phase in cell fraction separation, TRIZOL reagent is generally used for direct dissolution and extraction of corresponding chromatin RNA, but the method can result in loss of genomic DNA information. For this reason, the present invention has attempted to develop a novel extraction reagent for dissolving the precipitate so that RNA and genomic DNA in the precipitate phase can be extracted simultaneously into the liquid phase, and then separated and purified separately using a silica gel purification procedure. The lysate (4M guanidine thiocyanate, 1M Tris-HCl, pH=7.4) is prepared, and then the lysate and TRIZOL reagent are uniformly mixed according to a volume of 1:2 to prepare the nuclear DNA/RNA separation buffer. From example 3, it is known that RNA and genomic DNA in the precipitated fraction can be simultaneously and efficiently extracted and purified by using the nucleoplasm DNA/RNA separation buffer, wherein the RNA extraction flow is 16min, the genomic DNA extraction flow is 14min, and the OD 260/280,OD260/230 parameters of the RNA and genomic DNA are good, so that the purity requirement of downstream high-throughput sequencing can be met. Therefore, on the basis of not affecting the classical RNA cell component separation scheme, the invention develops a scheme for extracting the corresponding genome DNA in the RNA cell component precipitation phase, achieves the aim of separating and purifying all nucleic acids (DNA and RNA) from different cell components, provides another level of genome DNA information for separating the cell components, makes up the defect of RNA cell component separation experiments, and provides technical reference for researches in related fields.
In order to further clearly demonstrate the specific procedures for constructing the method of the present invention for separating, extracting and purifying nucleic acids suitable for different cellular components, detailed descriptions will be provided below in connection with examples 1 to 3.
EXAMPLE 1 RNA binding solution exploration of compatible cytoplasmic component lysates
1. The experimental procedure was as follows:
(1) As shown in table 1, corresponding RNA binding solutions (1-8M guanidine thiocyanate, 1M Tris-HCl, ph=7.4) were prepared;
(2) Preparing a cytoplasmic component simulation solution, namely a lysis buffer solution 1 (10 mM Tris-HCl, pH 7.5,0.05% Igepal 630,150 mM NaCl, adding 2 mu L of RNase inhibitor to each 1 mL) and a sucrose cushion lysis solution (the nuclease-free sucrose is prepared in the lysis buffer solution 1 according to the mass percentage of 24%), and then mixing the lysis buffer solution 1 and the sucrose cushion lysis solution according to the volume ratio of 1:2.5 to prepare the cytoplasmic component simulation solution, and performing RNA recovery simulation fumbling in the solution;
(3) 200 mu L of cytoplasmic component simulation solution and 200 mu L of RNA binding solution shown in Table 1 are taken, 830ng total RNA is added after the mixture is uniformly mixed, and then 400 mu L of absolute ethyl alcohol is added for uniform mixing;
(4) Directly incubating the mixture with a silica gel column for 1min, and centrifuging 12000g for 30s;
(5) 400. Mu.L of an RNA rinse 1 solution (80% ethanol, 2M guanidine hydrochloride) was added thereto, and the mixture was centrifuged at 12000g for 30s for rinsing;
(6) 700. Mu.L of RNA rinse solution 2 (80% ethanol) was added, the mixture was centrifuged for 30s for one time at 12000g, and the mixture was air-thrown for 1min and eluted with 12000g of pure water for 1min, which was a recovered RNA sample, and the sample was left for quality control.
2. Experimental results:
As can be seen from FIG. 1, the RNA extracted in this example has good quality, and the cytoplasmic component RNA extraction process does not lead to RNA degradation, and since the added RNA binding solution contains guanidine thiocyanate with different concentrations, guanidine thiocyanate has a certain inhibition effect on RNase, and RNA degradation caused by careless introduction of RNase pollution during RNA extraction and purification can be prevented. Meanwhile, as can be seen from Table 1, the recovery rate of the prepared 8 RNA binding solutions was between 72.3 and 87.8%, wherein the recovery rate of the RNA binding solution of 5M guanidine thiocyanate was the highest and reached 87.8%. In addition, OD 260/280 is greater than 1.8 and OD 260/230 is greater than 1.8, all meeting the purity requirements of high throughput sequencing. Thus, subsequent experiments will employ 5M guanidine thiocyanate, 1M Tris-HCl, ph=7.4 as cytoplasmic component RNA binding solution.
TABLE 1 extraction Effect of different RNA binding fluids on cytoplasmic component RNA
EXAMPLE 2 RNA binding solution compatible with Nuclear free component lysate
1. The experimental procedure was as follows:
(1) As shown in table 2, corresponding RNA binding solutions (1-8M guanidine thiocyanate, 1M Tris-HCl, ph=7.4) were prepared;
(2) Preparing a nuclear free component simulation solution, namely a glycerol buffer solution (20 mM Tris-HCl, pH 7.9;75 mM NaCl;0.5 mM EDTA;0.85 mM DTT;0.125 mM PMSF;50% glycerol) and a nuclear lysis buffer solution (10 mM HEPES,pH 7.6;1 mM DTT;7.5 mM MgCl2;0.2 mM EDTA;0.3M NaCl;1M urea; 1% Igepal 630), mixing the glycerol buffer solution and the nuclear lysis buffer solution according to the volume ratio of 1:1 to prepare the nuclear free component simulation solution, and performing RNA recovery simulation searching in the solution;
(3) 200 mu L of the nuclear free component simulation solution and 200 mu L of the RNA binding solution shown in Table 2 are taken, 830ng of total RNA is added after the mixture is uniformly mixed, and 400 mu L of absolute ethyl alcohol is added and uniformly mixed;
(4) Directly incubating the mixture with a silica gel column for 1min, and centrifuging 12000g for 30s;
(5) 400. Mu.L of an RNA rinse 1 solution (80% ethanol, 2M guanidine hydrochloride) was added thereto, and the mixture was centrifuged at 12000g for 30s for rinsing;
(6) 700. Mu.L of RNA rinse solution 2 (80% ethanol) was added, the mixture was centrifuged for 30s for one time at 12000g, and the mixture was air-thrown for 1min and eluted with 12000g of pure water for 1min, which was a recovered RNA sample, and the sample was left for quality control.
2. Experimental results:
As can be seen from fig. 2, the RNA extracted from the core free component RNA mimic was of good quality, without degradation, and the formulated RNA binding solution was compatible with the core free lysate. Meanwhile, it can be found from Table 2 that the recovery rates of the different RNA binding solutions were between 75.9% and 97.6%, with the guanidine thiocyanate concentrations of 6M and 7M reaching an ultra-high recovery rate of 97.5%. In addition, OD 260/280 is greater than 1.8 and OD 260/230 is greater than 1.8, all meeting the purity requirements of high throughput sequencing. Thus, subsequent experiments will employ 6M guanidine thiocyanate, 1M Tris-HCl, ph=7.4 as RNA binding solution for the nuclear free fraction.
TABLE 2 extraction Effect of different RNA binding fluids on nuclear free constituent RNAs
Example 3 development of nucleoplasmic DNA/RNA isolation buffer and application in separation of real cell fractions
1. The experimental procedure was as follows:
1.1, cell component separation experiments:
(1) 5X 10 6-1×107 human 293T cells were collected, washed with 1mL ice-cold (pre-chilled on ice, same below) PBS/1 mM EDTA buffer and cell pellet was collected by centrifugation at 500 g at 4 ℃;
(2) 200. Mu.L of ice-cold lysis buffer 1 (10 mM Tris-HCl, pH 7.5;0.05% Igepal 630;150 mM NaCl; 2. Mu.L RNase inhibitor per 1 mL) was added to the cell pellet, after incubation on ice for 5 minutes, the lysate was gently pipetted and mixed, and then covered onto a 2.5-fold volume ice-cold sucrose pad (24% by mass of nuclease-free sucrose in lysis buffer 1), centrifuged at 15,000 g for 10 minutes at 4℃to collect all supernatant as cytoplasmic fraction for use, and the remaining nuclear pellet was then subjected to the next step;
(3) 200. Mu.L ice-cold PBS/1 mM EDTA buffer was gently added to the nuclear pellet and PBS/EDTA was removed immediately after the pellet was not disturbed. The cell nucleus pellet was resuspended in 200. Mu.L of pre-chilled glycerol buffer (20 mM Tris-HCl, pH 7.9;75 mM NaCl;0.5 mM EDTA;0.85 mM DTT;0.125 mM PMSF;50% glycerol), gently mixed, and then added with an equal volume of ice-cold nuclear lysis buffer (10 mM HEPES,pH 7.6;1 mM DTT;7.5 mM MgCl 2; 0.2 mM EDTA;0.3M NaCl;1M urea; 1% Igepal 630), vortexed vigorously 2 times, 5 seconds each;
(4) After incubating the mixture on ice for 2 minutes, the supernatant was collected as a nuclear free fraction by centrifugation at 15,000 g for 2 minutes at 4℃followed by gently rinsing the pellet (without agitation) with ice-cold PBS/1 mM EDTA buffer and collecting the pellet as a chromosome-related fraction.
1.2, Cytoplasmic fraction RNA extraction:
For cytoplasmic fractions, RNA was extracted as in example 1, using 5M guanidine thiocyanate, 1M Tris-HCl, pH=7.4 as cytoplasmic fraction RNA binding solution.
1.3, Extracting nuclear free component RNA:
For the nuclear free fraction, RNA was extracted as in example 2, using 6M guanidine thiocyanate, 1M Tris-HCl, ph=7.4 as RNA binding solution for the nuclear free fraction.
1.4, Co-extraction of chromosomal component RNA and genomic DNA:
for chromosome Guan Zufen, the RNA and genomic DNA co-extraction procedure included the following steps:
(1) Preparing lysate (4M guanidine thiocyanate, 1M Tris-HCl, pH=7.4), and uniformly mixing the lysate and TRIZOL reagent according to a volume ratio of 1:2 (for example, 0.5mL lysate and 1mL TRIZOL) to prepare a nuclear DNA/RNA separation buffer;
(2) 1mL of nucleoplasm DNA/RNA separation buffer is used for dissolving all relevant components of the chromatin obtained by separation in the step 1.1, and gun blowing is used for carrying out the necessary thorough dissolution;
(3) 1/5 volume of chloroform, 200. Mu.L, was added and centrifuged at 12000g for 10min at 4 ℃;
(4) Incubating the supernatant with a silica gel column for 1min, and centrifuging 12000g for 30s;
(5) 700. Mu.L of DNA rinsing solution (80% ethanol+100 mM NaCl) was added, and the mixture was centrifuged for 30s once at 12000g, and the mixture was subjected to air-spinning for 1min and purified water for 1min, which was a sample of isolated genomic DNA, and the sample was left for quality control.
(6) Collecting the flow-through liquid in the step (4), adding absolute ethanol with the volume of 1 time, incubating with a new silica gel column for 1min, and centrifuging for 30s with 12000 g;
(7) 400. Mu.L of an RNA rinse 1 solution (80% ethanol, 2M guanidine hydrochloride) was added thereto, and the mixture was centrifuged at 12000g for 30s for rinsing;
(8) 700 mu L of RNA rinsing liquid 2 solution (80% ethanol) is added, the solution is centrifuged for 30s for one time with 12000g, 1min is thrown out, and 12000g pure water is used for eluting for 1min, so that a chromatin RNA sample is reserved for quality inspection.
2. Experimental results:
As can be seen from fig. 3 and table 3, in the actual application process of separating cell components, the extracted cytoplasmic component RNA, nuclear free component RNA and chromatin component RNA have good purification effect, no obvious degradation occurs, and simultaneously, OD 260/280 is greater than 1.8, OD 260/230 is greater than 1.8, which all meet the purity requirement of high-throughput sequencing. Wherein the chromatin component RNA exhibited a unique band of chromatin binding RNA, namely 45S precursor rRNA (asterisk indicates position), indicating that the cell component separation experiment and the corresponding extraction and purification experiment were successful. In addition, lane 4 is genomic DNA in a chromatin component, has good OD 260/280,OD260/230 parameters and high purity, can meet the purity requirement of high-throughput sequencing, and further proves that the nucleoplasm DNA/RNA separation buffer and the corresponding purification flow developed by the invention can simultaneously and efficiently co-extract and efficiently distinguish chromatin RNA from genomic DNA, which is the initiative of the invention.
TABLE 3 nucleic acid extraction Effect of different cell Components
In summary, the invention develops a method compatible with cell component fractionation and efficient purification of nucleic acid, so that the downstream can be compatible with the purity requirement of high-throughput sequencing, and specifically comprises an efficient purification flow of cytoplasmic component RNA, an efficient purification flow of nuclear free RNA and an efficient purification flow of nuclear DNA/RNA. The invention improves the dissolution extraction method of chromatin components (precipitation components), develops a nucleoplasm DNA/RNA separation buffer, and is compatible with the efficient separation and purification flow of DNA and RNA silica gel.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (5)
1. A method for separating, extracting and purifying nucleic acid from different cell components is characterized by adopting a kit, wherein the kit comprises an RNA binding solution, absolute ethyl alcohol, an RNA rinsing solution 1 solution, an RNA rinsing solution 2 solution, a nucleoplasm DNA/RNA separation buffer, chloroform and a DNA rinsing solution, the RNA binding solution consists of 1-8M guanidine thiocyanate and 1M Tris-HCl, the pH=7.4, the RNA rinsing solution 1 solution consists of 80% ethyl alcohol and 2M guanidine hydrochloride, the RNA rinsing solution 2 solution is 80% ethyl alcohol solution, the nucleoplasm DNA/RNA separation buffer consists of a lysis solution and a TRIZOL reagent according to the volume ratio of 1:2, the lysis solution consists of 4M guanidine thiocyanate and 1M Tris-HCl, the pH=7.4, the DNA rinsing solution consists of 80% ethyl alcohol and 100mM NaCl, and the method comprises the following steps:
S1, separating cytoplasmic components, nuclear free components and chromosome components from cells;
S2, extracting RNA of cytoplasmic components or nuclear free components:
s21, adding RNA binding solution into cytoplasmic components or nuclear free components, and then adding absolute ethyl alcohol to mix uniformly, wherein the volume ratio of the cytoplasmic components or the nuclear free components, the RNA binding solution and the absolute ethyl alcohol is 1:1:2-4;
s22, incubating the mixed solution of the S21 with a silica gel column to combine RNA with the silica gel column;
S23, adding the RNA rinsing liquid 1 solution into the silica gel column of the S22 for rinsing;
S24, adding an RNA rinsing liquid 2 solution into the silica gel column of S23 for rinsing, and then performing air-throwing and pure water elution to obtain an RNA sample;
S3, co-extraction of chromatin-associated RNA and genomic DNA:
s31, separating buffer by using nucleoplasm DNA/RNA to dissolve chromosome components, adding chloroform, centrifuging, mixing uniformly and layering;
s32, taking supernatant in the step S31, and incubating with a silica gel column to combine DNA with the silica gel column;
S33, adding a DNA rinsing liquid into the silica gel column of S32 for rinsing, and then performing air-throwing and pure water elution to obtain a genome DNA sample;
S34, collecting the flow-through liquid of S32, adding absolute ethyl alcohol, then incubating the flow-through liquid with a new silica gel column, enabling RNA to be combined with the silica gel column according to the volume ratio of the flow-through liquid to the absolute ethyl alcohol of 1:1-2, and then processing according to S23 and S24 to obtain a chromatin RNA sample.
2. The method according to claim 1, wherein in S21, the RNA-binding solution added to the cytoplasmic fraction consists of 5M guanidine thiocyanate and 1M Tris-HCl, pH=7.4, and the RNA-binding solution added to the nuclear free fraction consists of 6-7M guanidine thiocyanate and 1M Tris-HCl, pH=7.4.
3. The method for separating, extracting and purifying nucleic acid from different cell fractions according to claim 1, wherein in S31, the volume ratio of the nucleic DNA/RNA separating buffer to chloroform is 4-6:1.
4. The method for separating and purifying nucleic acid from different cell components according to claim 1, wherein the step of separating the cytoplasmic fraction, the nuclear free fraction and the chromosomal fraction from the cell by S1 comprises the steps of:
S11, washing cells with ice-cold PBS/1 mM EDTA buffer solution, and centrifuging to collect cell sediment;
S12, adding ice-cold lysis buffer 1 into the cell sediment of S11, incubating on ice, slightly pipetting, uniformly mixing the lysis solution, covering the lysis solution on an ice-cold sucrose cushion, centrifuging, collecting the supernatant as a cytoplasmic component for later use, and allowing the rest cell nucleus sediment to enter the next step, wherein the lysis buffer 1 consists of 10mM Tris-HCl,0.05% IGEPAL CA-630 and 150 mM NaCl, 2 mu L of RNase inhibitor is added per 1mL, the pH=7.5, and the sucrose cushion is prepared by dissolving the nuclease-free sucrose in the lysis buffer 1 according to the mass percentage of 20-30%;
S13, adding ice-cold PBS/1 mM EDTA buffer solution into the cell nucleus precipitate obtained in S12, removing PBS/EDTA after not disturbing the precipitate, re-suspending the cell nucleus precipitate in precooled glycerol buffer solution, adding an equal volume of ice-cold nucleus lysis buffer solution after uniformly mixing, incubating, centrifuging to collect supernatant as a free component of the nucleus for later use, flushing the precipitate with the ice-cold PBS/1 mM EDTA buffer solution, and collecting the precipitate as a related component of the chromosome, wherein the glycerol buffer solution consists of 20 mM Tris-HCl,75 mM NaCl,0.5 mM EDTA,0.85 mM DTT,0.125 mM PMSF,50% glycerol, the pH=7.9, and the nucleus lysis buffer solution consists of 10mM HEPES,1 mM DTT,7.5 mM MgCl 2, 0.2 mM EDTA,0.3M NaCl,1M urea and 1% IGEPAL CA-630, and the pH=7.6.
5. The method according to claim 4, wherein 1. 1 mL ice-cold PBS/1. 1mM EDTA buffer is added to each of 5X 10 6-1×107 cells in S11.
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