A method for separating, extracting and purifying nucleic acids from different cell components

Abstract

The present invention belongs to the field of nucleic acid extraction technology, and specifically relates to a method for separating, extracting and purifying nucleic acids from different cell components. The present invention develops a separation and purification method for liquid-phase RNA such as cytoplasmic components and nuclear free components, so that the extracted RNA can be compatible with the highest standards of nucleic acid mass spectrometry and high-throughput sequencing, and makes the operation process simpler, takes less time, has lower costs, and has better and more efficient extraction effects; at the same time, on this basis, a corresponding nuclear cytoplasmic DNA / RNA separation buffer is also developed, and an efficient nucleic acid purification process is provided, so that while obtaining high-quality chromatin RNA, genomic DNA can also be extracted intact, high-quality and high-purity, providing another level of information for the classic component separation scheme of RNA dynamic biology, namely genomic DNA information, thereby enabling all nucleic acid information of various cell components to be efficiently separated and purified.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid extraction, and in particular relates to a method for separating, extracting and purifying nucleic acids from different cell components. Background Art

[0002] The separation of cellular components of nucleic acids, especially RNA, is of great significance for studying the function of RNA. In eukaryotic cells, the synthesis, processing and function of RNA are carried out in specific subcellular compartments. By fractionating the biochemical components of cells, the different stages of the RNA life cycle can be deeply analyzed. At present, cell component separation technology is widely used in the study of the synthesis, processing and transport of biological molecules and organelles; in recent years, this technology has been combined with new deep sequencing technology to provide unprecedented insights into the generation and function of primary RNA transcripts.

[0003] The basic principle and operation process of RNA cell component separation are as follows: first, cells are lysed by mild detergents to release organelles and other cell components into the suspension while maintaining the integrity of the nuclear membrane. The cell nuclei are initially purified by centrifugation and precipitation with 24% sucrose, and the supernatant is further centrifuged at high speed to obtain cytoplasmic extracts. The second step of component separation uses a method developed by Wuarin and Schibler in 1994, which aims to separate chromatin and nucleoplasm complexed with nascent RNA to study whether RNA splicing co-occurs with transcription. Specifically, the method involves gently resuspending the precipitated nuclei in a buffer containing 50% glycerol and lysing them by quickly adding 1% Igepal 630 and 1 M urea. The key innovation is to use urea instead of ionic detergents to destroy the nuclear membrane, which can maintain the binding of histones including histone H1 to genomic DNA, retain the compact chromatin structure, and allow the chromatin-RNA complex to be centrifuged and precipitated by a desktop centrifuge. Finally, RNA can be recovered from each separated component by phenol-chloroform extraction or TRIZOL extraction. At present, this method has been optimized by many laboratories and applied to the study of nascent transcription, exon usage, splicing dynamics, RNA processing, transport and degradation, especially in capturing short-lived chromatin-related primary transcripts (such as microRNA precursors). Due to its simple operation and applicability to a variety of cell types, this technology has important value and wide applicability in studying the dynamic biological process of RNA.

[0004] At present, in terms of RNA extraction after component separation, cytoplasmic component RNA exists in cytoplasmic lysate, nuclear free RNA components also exist in liquid lysate, and chromatin RNA (precipitation phase) can be directly dissolved with TRIZOL and purified and separated using the classic TRIZOL RNA extraction method. As for RNA (cytoplasmic component and nuclear free component RNA) present in liquid lysate, due to its instability and easy degradation, it is necessary to purchase TRIZOL-LS reagent for liquid phase extraction. 0.25mL liquid phase solution requires 0.75mL TRIZOL-LS reagent. On the one hand, TRIZOL-LS is expensive, and on the other hand, the volume after TRIZOL-LS treatment is huge, which is not conducive to the subsequent separation and purification of RNA. At the same time, this RNA cell component separation method often leads to the loss of genomic DNA information of the cell tissue due to the use of a special reagent that maintains chromatin structure (aimed at isolating the real chromatin-bound RNA) and TRIZOL extraction. Therefore, it is urgent to develop new methods for separating RNA cell components so that genomic DNA can be extracted simultaneously during the separation of chromatin RNA precipitation, which can not only provide transcriptome information of each cell component, but also provide its corresponding genome information.

[0005] In our previously disclosed patent CN118240814A, a principle and method for distinguishing DNA and RNA in a liquid phase in vitro was established, but this method is only applicable to the liquid phase in vitro and has not been applied to real cell samples. Therefore, it is questionable whether it can also distinguish DNA and RNA in actual cell samples and chromosome cell sediments that have undergone complex processing. Moreover, one of the most important problems is that the DNA and RNA in complex cell samples or cell sediments are often not present in the aqueous phase at the same time, so in principle it cannot be applied to actual cell samples. For example, after the chromatin precipitation phase of the traditional classic cell component separation is treated with a unique extraction reagent that makes the chromatin structure compact, the chromatin RNA and DNA are present in the compact cell sediment, and the RNA therein can still be redissolved in the liquid phase by TRIZOL, but the DNA in the cell sediment phase cannot be redissolved in the liquid phase after using the TRIZOL reagent, so it is impossible to prepare high-quality and high-purity DNA. Therefore, it is urgent to develop a DNA / RNA extraction reagent so that both RNA and DNA in the cell sediment phase can be efficiently redissolved in the liquid phase to facilitate the subsequent silica gel purification process.

[0006] In summary, for RNA components present in liquid lysate, the traditional separation and purification process is more than 1h, and the separation reagents are expensive, and the volume is huge after adding the separation reagents, and the experimental operation is cumbersome. RNA in liquid lysate is also easy to degrade, so it is urgent to develop a method for rapid separation and purification of RNA that is compatible and adaptable to liquid lysate. At the same time, in the traditional classic RNA cell component separation scheme, genomic DNA information is lost, and this problem has not been solved so far. If the corresponding genomic DNA can be extracted and separated in the RNA cell component separation scheme, the nucleic acid (RNA and DNA) components in each cell component can be basically separated and purified, which is conducive to obtaining complete genomic information and transcriptome information. In addition, although our previous patent CN118240814A established a method for efficient separation and purification of DNA and RNA, it is only for DNA and RNA present in the in vitro liquid phase, and it is still powerless for RNA and DNA in the chromatin precipitation phase. Therefore, it is necessary to develop a separation and purification method that is suitable for liquid or precipitated RNA such as cytoplasmic components, nuclear free components and chromatin, while making the operation process simpler, time-saving, cost-effective, and extraction more effective and efficient. Summary of the invention

[0007] In order to solve the problems of complex operation, time-consuming, high cost, easy RNA degradation, etc. in the separation and purification of liquid-phase RNA such as cytoplasmic components and nuclear free components in the classic RNA cell component separation scheme, the present invention develops a separation and purification method for liquid-phase RNA such as cytoplasmic components and nuclear free components, wherein the recovery rate of cytoplasmic RNA is between 72.3% and 87.8%, and the recovery rate of nuclear free RNA is between 75.9% and 97.6%, and the purity is high, and its OD 260 / 280 (greater than 1.8), OD 260 / 230 (greater than 1.8) parameters are good and compatible with the highest standards of nucleic acid mass spectrometry and high-throughput sequencing. At the same time, the purification time of cytoplasmic RNA and nuclear free RNA is only 4 minutes, and the purification time of chromatin RNA is 16 minutes. The operation is simple and the duration is short, which can meet the preparation needs of high-throughput samples. In addition, for the DNA in the chromatin precipitation component, a corresponding nucleoplasmic DNA / RNA separation buffer has been developed. This buffer can efficiently separate and extract chromatin RNA and genomic DNA at the same time, and the purification time of genomic DNA is 14 minutes. Therefore, the scheme of the present invention can basically efficiently grade and separate all nucleic acid components in various components of the cell, providing technical support for the research field of RNA dynamic biology.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides a kit for separating, extracting and purifying nucleic acids from different cell components, the kit comprising RNA binding solution, anhydrous ethanol, RNA rinsing solution 1 solution, RNA rinsing solution 2 solution, nuclear cytoplasmic DNA / RNA separation buffer, chloroform, and DNA rinsing solution;

[0010] The RNA binding solution consists of 1-8M guanidine thiocyanate and 1M Tris-HCl, with a pH of 7.4; the RNA rinsing solution 1 consists of ethanol and guanidine hydrochloride; the RNA rinsing solution 2 is an ethanol solution; the nucleoplasmic DNA / RNA separation buffer consists of a lysis solution and TRIZOL reagent, the lysis solution consists of 4M guanidine thiocyanate and 1M Tris-HCl, with a pH of 7.4; the DNA rinsing solution consists of ethanol and NaCl.

[0011] Preferably, the RNA rinse solution 1 is composed of 80% ethanol and 2M guanidine hydrochloride, the RNA rinse solution 2 is an 80% ethanol solution, the nucleocytoplasmic DNA / RNA separation buffer is composed of a lysis solution and a TRIZOL reagent in a volume ratio of 1:2, and the DNA rinse solution is composed of 80% ethanol and 100mM NaCl.

[0012] The second aspect of the present invention provides a method for separating, extracting and purifying nucleic acids from different cell components, specifically using the kit described in the first aspect to separate, extract and purify nucleic acids from different cell components, comprising the following steps:

[0013] S1. Separate cytoplasmic components, nuclear free components and chromosome components from cells;

[0014] S2. Extraction of RNA from cytoplasmic fractions or nuclear free fractions:

[0015] S21, adding RNA binding solution to the cytoplasmic fraction or the nuclear free fraction, and then adding anhydrous ethanol to mix;

[0016] S22, incubating the mixed solution of S21 with the silica gel column to allow the RNA to bind to the silica gel column;

[0017] S23, adding RNA rinsing solution 1 solution to the silica gel column in S22 for rinsing;

[0018] S24, adding RNA rinsing solution 2 to the silica gel column of S23 for rinsing, and then spinning and eluting with pure water to obtain an RNA sample.

[0019] S3. Co-extraction of chromatin-associated RNA and genomic DNA:

[0020] S31, dissolve the chromosome components with nuclear DNA / RNA separation buffer, then add chloroform, centrifuge to mix and separate the layers;

[0021] S32, taking the supernatant in S31 and incubating it with a silica gel column to allow the DNA to bind to the silica gel column;

[0022] S33, adding DNA rinsing solution to the silica gel column in S32 for rinsing, and then spinning and eluting with pure water to obtain a genomic DNA sample;

[0023] S33. Collect the flow-through of S32, add anhydrous ethanol and incubate it with a new silica gel column to allow the RNA to bind to the silica gel column, and then process it according to S23 and S24 to obtain a chromatin RNA sample.

[0024] The present invention has developed a liquid-phase RNA separation and purification method suitable for cytoplasmic components and nuclear free components, making the operation process simpler, shorter, lower cost, better extraction effect and more efficient. At the same time, a nuclear cytoplasmic DNA / RNA separation buffer has also been developed, and an efficient nucleic acid purification process is also matched, so that while obtaining high-quality chromatin RNA, the genomic DNA can also be extracted intact, high-quality and high-purity, providing another level of information for the classic component separation scheme of RNA dynamic biology, namely, genomic DNA information, so that all nucleic acid information of each component of the cell can be efficiently separated and purified.

[0025] Preferably, in S21, the RNA binding solution added to the cytoplasmic component consists of 5M guanidine thiocyanate and 1M Tris-HCl, with a pH of 7.4; the RNA binding solution added to the nuclear free component consists of 6-7M guanidine thiocyanate and 1M Tris-HCl, with a pH of 7.4.

[0026] Preferably, in S21, the volume ratio of the cytoplasmic component or the nuclear free component, the RNA binding solution and the anhydrous ethanol is 1:1:2-4.

[0027] Preferably, in S31, the volume ratio of nuclear-cytoplasmic DNA / RNA separation buffer to chloroform is 4-6:1.

[0028] Preferably, in S33, the volume ratio of the flow-through liquid to anhydrous ethanol is 1:1-2.

[0029] Preferably, in S33, after adding the DNA rinsing solution, rinse at 10000-20000 g for 30 s-1 min, spin for 1-2 min, and elute with 10000-20000 g pure water for 1-3 min.

[0030] Preferably, in S24, after adding RNA rinsing solution 2, rinse at 10000-20000 g for 30 s-1 min, spin for 1-2 min, and elute with pure water at 10000-20000 g for 1-3 min.

[0031] Preferably, in S22, S32, and S33, after incubation with the silica gel column, centrifugation is performed at 10,000-20,000 g for 30 seconds to 1 minute to allow the RNA / DNA to bind to the silica gel column.

[0032] Preferably, in S23, after adding RNA rinsing solution 1, rinse at 10000-20000g for 30s-1min.

[0033] Preferably, S1 separates the cytoplasmic components, the nuclear free components and the chromosomal components from the cells and specifically comprises the following steps:

[0034] S11, wash the cells with ice-cold PBS / 1 mM EDTA buffer, and collect the cell pellet by centrifugation;

[0035] S12, add ice-cold lysis buffer 1 to the cell pellet of S11, incubate on ice, gently pipette to mix the lysis solution, then cover it on an ice-cold sucrose cushion, centrifuge and collect the supernatant as a cytoplasmic component for standby use, and the remaining cell nuclear pellet proceeds to the next step; the lysis buffer 1 is composed of 10 mM Tris-HCl, 0.05% IGEPAL CA-630, 150 mM NaCl, 2 μL RNase inhibitor is added per 1 mL, pH=7.5; the sucrose cushion is prepared by dissolving nuclease-free sucrose in lysis buffer 1 at a mass percentage of 20-30%;

[0036] S13. Add ice-cold PBS / 1 mM EDTA buffer to the cell nuclear pellet obtained in S12, remove PBS / EDTA without disturbing the pellet, resuspend the cell nuclear pellet in pre-cooled glycerol buffer, mix well, add an equal volume of ice-cold nuclear lysis buffer, mix well and incubate, then centrifuge to collect the supernatant as the nuclear free component for stand-alone use, and rinse the pellet with ice-cold PBS / 1 mM EDTA buffer, and collect the pellet as the chromosome-related component; the glycerol buffer consists of 20 mM Tris-HCl, 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, 0.125 mM PMSF, 50% glycerol, pH=7.9; the nuclear lysis buffer consists of 10 mM HEPES, 1 mM DTT, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M urea, 1% IGEPAL CA-630, pH=7.6.

[0037] More preferably, in S11, every 5×10 6 -1×10 7 Add 1 mL of ice-cold PBS / 1 mM EDTA buffer to each cell.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention develops a separation and purification method for liquid-phase RNA such as cytoplasmic components and nuclear free components, so that the extracted RNA is compatible with the highest standards of nucleic acid mass spectrometry and high-throughput sequencing, and makes the operation process simpler, the time is shorter, the cost is lower, the extraction effect is better and more efficient; at the same time, on this basis, a corresponding nuclear cytoplasmic DNA / RNA separation buffer is also developed, and the buffer can simultaneously separate and extract chromatin RNA and genomic DNA, so that all nucleic acid information of each component of the cell can be efficiently separated and purified.

[0040] The present invention has developed a method for efficient RNA purification that is compatible with the classical RNA cell component separation experiment, and provides a process for efficient purification of cytoplasmic RNA components and a method for efficient purification of nuclear free RNA components. The entire RNA extraction and purification time is about 4 minutes, the RNA purity is high, and it is applicable to the highest standards of high-throughput sequencing. At the same time, a nuclear DNA / RNA separation buffer has been developed to achieve the simultaneous extraction of DNA and RNA components from chromatin precipitation, and the integrity and purity of DNA and RNA both meet the highest standards, and are also compatible with subsequent high-throughput sequencing requirements. Among them, the attempt to separate DNA from chromatin precipitation is the first, and the entire DNA and RNA purification time is 14 minutes for DNA and 16 minutes for RNA, which greatly improves the classical scheme for RNA component separation and provides technical support for the development of the field of RNA dynamic biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The extraction effects of different RNA binding solutions on cytoplasmic component RNA (lanes 1-8 represent the experimental treatments of different RNA binding solutions, see Table 1 for details, M is Marker);

[0042] Figure 2 The extraction effects of different RNA binding solutions on the free nuclear component RNA (lanes 1-8 represent the experimental treatments of different RNA binding solutions, see Table 2 for details, M is a marker);

[0043] Figure 3 It is the nucleic acid extraction effect of different cell components (lanes 1-4 represent RNA / DNA of different cell components, see Table 3 for details, M is Marker).

[0044] Figures 1 to 3 In the figure, M is Marker, and the sizes from large to small are 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp. DETAILED DESCRIPTION

[0045] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0046] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0047] The purification column used in the present invention is from Zymo research, model C1004-50, and any silica gel column with guaranteed quality on the market meets the requirements.

[0048] The present invention aims at the problem that it is difficult to extract and purify RNA in liquid phase after fractionation and separation of RNA cell components, and develops a silica gel process scheme that is highly efficient and compatible with liquid phase RNA extraction. Meanwhile, the present invention can also provide a reference for the establishment of other efficient fractionation and separation schemes for nucleic acids present in liquid phase lysates.

[0049] In the classic RNA cell component separation scheme, the cytoplasmic component and the nuclear free component are present in the liquid lysate, and TRIZOL-LS reagent is usually used for subsequent treatment. On the one hand, TRIZOL-LS is expensive, and on the other hand, one volume of the liquid lysate needs to add 3 volumes of TRIZOL-LS reagent, and the solution volume is huge, which makes the subsequent extraction and purification very cumbersome. To this end, the present invention is based on the principle of RNA silica gel extraction (RNA can be combined with silica gel column and separated under the action of 1-2 times of anhydrous ethanol in a certain salt solution), starting from solving the compatibility problem between RNA cell component separation lysate and silica gel column system, and exploring efficient nucleic acid separation and purification schemes. First, the corresponding RNA binding solution is developed. At the same time, considering that the components of the cytoplasmic lysate and the nuclear free lysate are already very complex, in order to facilitate the subsequent silica gel purification and reduce the reagent cost, the formula of the RNA binding solution is further simplified. Finally, it is found that only the single salt component of guanidine thiocyanate can achieve the compatibility of the cytoplasmic lysate and the nuclear free lysate with the silica gel column. As can be seen from Examples 1 and 2, the present invention can achieve a recovery rate of 72.3% to 87.8% of RNA in cytoplasmic lysate using only a simple RNA binding solution, i.e., 1-8M guanidine thiocyanate, 1M Tris-HCl, pH=7.4, and a recovery rate of 75.9% to 97.6% in nuclear free lysate, and the isolated RNA has a high purity, OD 260 / 280 Greater than 1.8, OD 260 / 230 Greater than 1.8, it can meet the purity requirements of high-throughput sequencing.

[0050] As for the precipitate phase in the separation of cell components, TRIZOL reagent is currently usually used to directly dissolve and extract the corresponding chromatin RNA, but this method will lead to the loss of genomic DNA information. To this end, the present invention attempts to develop a new extraction reagent for dissolving the precipitate, so that the RNA and genomic DNA in the precipitate phase can be extracted into the liquid phase at the same time, and then use the silica gel purification process to separate and purify them separately. That is, first prepare a lysis solution (4M guanidine thiocyanate, 1M Tris-HCl, PH=7.4), and then mix it with the TRIZOL reagent in a volume of 1:2 to prepare a nuclear DNA / RNA separation buffer. It can be seen from Example 3 that the use of this nuclear DNA / RNA separation buffer can efficiently extract and purify the RNA and genomic DNA in the precipitate component at the same time, wherein the RNA extraction process is 16min, the genomic DNA extraction process is 14min, and the OD of the two is 2. 260 / 280 , O.D. 260 / 230 The parameters are good and can meet the purity requirements of downstream high-throughput sequencing. Therefore, the present invention has developed a scheme for extracting the corresponding genomic DNA in the RNA cell component precipitation phase without affecting the classic RNA cell component separation scheme, and achieved the purpose of separating and purifying all nucleic acids (DNA and RNA) from different cell components, providing another level of genomic DNA information for the separation of cell components, making up for the shortcomings of RNA cell component separation experiments, and providing technical reference for research in related fields.

[0051] In order to further clearly present the specific process of constructing the method for separating, extracting and purifying nucleic acids applicable to different cell components of the present invention, a detailed description is given below in conjunction with Examples 1-3.

[0052] Example 1: Exploration of RNA binding solutions compatible with cytoplasmic component lysates

[0053] 1. The experimental operation is as follows:

[0054] (1) Prepare the corresponding RNA binding solution (1-8 M guanidine thiocyanate, 1 M Tris-HCl, pH = 7.4) as shown in Table 1;

[0055] (2) Prepare a cytoplasmic component simulation solution, namely, lysis buffer 1 (10 mM Tris-HCl, pH 7.5, 0.05% Igepal 630, 150 mM NaCl, 2 μL RNase inhibitor per 1 mL) and sucrose cushion lysis solution (24% by mass of nuclease-free sucrose is prepared in lysis buffer 1), and then mix lysis buffer 1 and sucrose cushion lysis solution in a volume ratio of 1:2.5 to prepare a cytoplasmic component simulation solution, and simulate RNA recovery in this solution;

[0056] (3) Take 200 μL of cytoplasmic component simulation solution + 200 μL of RNA binding solution described in Table 1, mix well, add 830 ng total RNA, then add 400 μL of anhydrous ethanol and mix well;

[0057] (4) Incubate the mixture directly with the silica gel column for 1 min and centrifuge at 12000g for 30 s;

[0058] (5) Add 400 μL of RNA washing solution 1 (80% ethanol, 2 M guanidine hydrochloride) and centrifuge at 12,000 g for 30 seconds for washing;

[0059] (6) Add 700 μL of RNA rinse solution 2 (80% ethanol) and centrifuge at 12,000 g for 30 s to rinse once; spin at room temperature for 1 min, and elute with pure water at 12,000 g for 1 min. This is the recovered RNA sample, which should be retained for quality inspection.

[0060] 2. Experimental results:

[0061] Depend on Figure 1 It can be seen that the RNA extracted in this example is of good quality, and the cytoplasmic component RNA extraction process will not cause RNA degradation. Since the added RNA binding solution contains different concentrations of guanidine thiocyanate, guanidine thiocyanate has a certain inhibitory effect on RNase, which can prevent RNA degradation caused by the accidental introduction of RNase contamination during RNA extraction and purification. At the same time, it can be found from Table 1 that the recovery rates of the 8 configured RNA binding solutions are between 72.3% and 87.8%, among which the RNA binding solution with 5M guanidine thiocyanate has the highest recovery rate, reaching 87.8%. In addition, OD 260 / 280 Greater than 1.8, OD 260 / 230 Greater than 1.8, all meet the purity requirements of high-throughput sequencing. Therefore, subsequent experiments will use 5M guanidine thiocyanate, 1M Tris-HCl, pH=7.4 as the cytoplasmic component RNA binding solution.

[0062] Table 1 The extraction effects of different RNA binding solutions on cytoplasmic RNA

[0063]

[0064] Example 2: Exploration of RNA binding solution compatible with nuclear free component lysis solution

[0065] 1. The experimental operation is as follows:

[0066] (1) Prepare the corresponding RNA binding solution (1-8 M guanidine thiocyanate, 1 M Tris-HCl, pH = 7.4) as shown in Table 2;

[0067] (2) Prepare a nuclear free component simulation solution, namely, 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) and nuclear lysis buffer (10 mM HEPES, pH 7.6; 1 mM DTT; 7.5 mM MgCl2; 0.2 mM EDTA; 0.3 M NaCl; 1 M urea; 1% Igepal630), then mix the glycerol buffer and nuclear lysis buffer in a 1:1 volume ratio to prepare a nuclear free component simulation solution, and simulate the RNA recovery in this solution;

[0068] (3) Take 200 μL of nuclear free component simulation solution + 200 μL of RNA binding solution described in Table 2, mix well, add 830 ng total RNA, then add 400 μL of anhydrous ethanol and mix well;

[0069] (4) Incubate the mixture directly with the silica gel column for 1 min and centrifuge at 12000g for 30 s;

[0070] (5) Add 400 μL of RNA washing solution 1 (80% ethanol, 2 M guanidine hydrochloride) and centrifuge at 12,000 g for 30 seconds for washing;

[0071] (6) Add 700 μL of RNA rinse solution 2 (80% ethanol) and centrifuge at 12,000 g for 30 s to rinse once; spin at room temperature for 1 min, and elute with pure water at 12,000 g for 1 min. This is the recovered RNA sample, which should be retained for quality inspection.

[0072] 2. Experimental results:

[0073] Depend on Figure 2 It can be seen that the RNA extracted by the nuclear free component RNA simulation is of good quality and has not been degraded, and the configured RNA binding solution is compatible with the nuclear free lysis solution. At the same time, it can be found from Table 2 that the recovery rates of different RNA binding solutions are between 75.9% and 97.6%, among which the 6M and 7M guanidine thiocyanate concentrations have reached an ultra-high recovery rate of 97.5%. In addition, OD 260 / 280 Greater than 1.8, OD 260 / 230 Greater than 1.8, all meet the purity requirements of high-throughput sequencing. Therefore, subsequent experiments will use 6M guanidine thiocyanate, 1M Tris-HCl, pH = 7.4 as the RNA binding solution for nuclear free components.

[0074] Table 2 The extraction effect of different RNA binding solutions on the free nuclear fraction RNA

[0075]

[0076] Example 3: Development of nuclear and cytoplasmic DNA / RNA separation buffer and its application in real cell component separation samples

[0077] 1. The experimental operation is as follows:

[0078] 1.1. Cell component separation experiment:

[0079] (1) Collect 5×10 6 -1×10 7 For individual 293T cells, wash with 1 mL of ice-cold PBS / 1 mM EDTA buffer and centrifuge at 500 g at 4°C to collect the cell pellet;

[0080] (2) Add 200 μL of ice-cold lysis buffer 1 (10 mM Tris-HCl, pH 7.5; 0.05% Igepal 630; 150 mM NaCl; 2 μL RNase inhibitor per 1 mL) to the cell pellet. After incubation on ice for 5 minutes, gently pipette to mix the lysate, then overlay it on a 2.5-fold volume of ice-cold sucrose cushion (24% mass percentage of nuclease-free sucrose is prepared in lysis buffer 1). Centrifuge at 15,000 g for 10 minutes at 4 °C. Collect all the supernatant as the cytoplasmic fraction for later use, and proceed to the next step for the remaining nuclear pellet.

[0081] (3) Gently add 200 μL of ice-cold PBS / 1 mM EDTA buffer to the nuclear pellet, and immediately remove the PBS / EDTA without disturbing the pellet. Resuspend the nuclear pellet in 200 μL 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 mix, and then add an equal volume of ice-cold nuclear lysis buffer (10 mM HEPES, pH 7.6; 1 mM DTT; 7.5 mM MgCl2; 0.2 mM EDTA; 0.3 M NaCl; 1 M urea; 1% Igepal 630), and vortex vigorously twice, 5 seconds each time;

[0082] (4) After incubating the mixture on ice for 2 minutes, centrifuge it at 15,000 g for 2 minutes at 4 °C and collect the supernatant as the nuclear-free fraction. Then, gently rinse the pellet with ice-cold PBS / 1 mM EDTA buffer (without disturbing it) and collect the pellet as the chromosome-associated fraction.

[0083] 1.2. Extraction of RNA from cytoplasmic components:

[0084] For the cytoplasmic component, RNA was extracted according to the protocol of Example 1, and 5 M guanidine thiocyanate, 1 M Tris-HCl, pH = 7.4 was used as the cytoplasmic component RNA binding solution.

[0085] 1.3. Extraction of RNA from nuclear free fractions:

[0086] For the nuclear free fraction, RNA was extracted according to the scheme of Example 2, and 6M guanidine thiocyanate, 1M Tris-HCl, pH=7.4 was used as the RNA binding solution for the nuclear free fraction.

[0087] 1.4. Co-extraction of chromosome component RNA and genomic DNA:

[0088] For chromosome-associated fractions, the RNA and genomic DNA co-extraction process includes the following steps:

[0089] (1) Prepare lysis buffer (4 M guanidine thiocyanate, 1 M Tris-HCl, pH = 7.4), and mix it with TRIZOL reagent in a volume ratio of 1:2 (for example, 0.5 mL lysis buffer plus 1 mL TRIZOL) to make nuclear and cytoplasmic DNA / RNA separation buffer;

[0090] (2) Use 1 mL of nuclear DNA / RNA separation buffer to dissolve all chromatin-related components separated in step 1.1. Use a pipette to make sure that they are completely dissolved.

[0091] (3) Add 1 / 5 volume of chloroform, i.e. 200 μL, and centrifuge at 12,000 g for 10 min at 4 °C;

[0092] (4) Incubate the supernatant with the silica gel column for 1 min and centrifuge at 12000g for 30 s;

[0093] (5) Add 700 μL of DNA washing solution (80% ethanol + 100 mM NaCl) and rinse once by centrifugation at 12,000 g for 30 s; spin for 1 min and elute with pure water at 12,000 g for 1 min. This is the separated genomic DNA sample, which should be retained for quality inspection.

[0094] (6) Collect the flow-through from step (4), add 1 volume of anhydrous ethanol, incubate with a new silica gel column for 1 min, and centrifuge at 12000g for 30 s;

[0095] (7) Add 400 μL of RNA washing solution 1 (80% ethanol, 2 M guanidine hydrochloride) and centrifuge at 12,000 g for 30 s for washing;

[0096] (8) Add 700 μL of RNA rinse solution 2 (80% ethanol) and centrifuge at 12,000 g for 30 seconds to rinse once; spin at room temperature for 1 minute and elute with pure water at 12,000 g for 1 minute. This is the chromatin RNA sample and should be retained for quality inspection.

[0097] 2. Experimental results:

[0098] Depend on Figure 3 As shown in Table 3, in the actual cell component separation application process, the extracted cytoplasmic component RNA, nuclear free component RNA, and chromatin component RNA all had good purification effects without obvious degradation. 260 / 280 Greater than 1.8, OD 260 / 230 The OD value of the chromatin component RNA is greater than 1.8, which meets the purity requirements of high-throughput sequencing. Among them, the chromatin component RNA has a band unique to chromatin-bound RNA, namely 45S pre-rRNA (the asterisk indicates the position), indicating that the cell component separation experiment and the corresponding extraction and purification experiment are successful. In addition, lane 4 is the genomic DNA in the chromatin component, and the OD value of the chromatin component RNA is greater than 1.8, which meets the purity requirements of high-throughput sequencing. 260 / 280 , O.D. 260 / 230 The parameters are good and the purity is high, which can meet the purity requirements of high-throughput sequencing, further proving that the nuclear DNA / RNA separation buffer and the corresponding purification process developed by the present invention can simultaneously and efficiently co-extract chromatin RNA and genomic DNA and efficiently distinguish them, which is also the first of the present invention.

[0099] Table 3 Nucleic acid extraction effects of different cell components

[0100]

[0101] In summary, the present invention has developed a method that is compatible with the fractionation and separation of cell components and the efficient purification of nucleic acids, so that its downstream is compatible with the purity requirements of high-throughput sequencing, specifically including the efficient purification process of cytoplasmic component RNA, the efficient purification process of nuclear free RNA, and the efficient purification process of nucleoplasmic DNA / RNA. The present invention improves the dissolution and extraction method of chromatin components (precipitation components), and develops a nucleoplasmic DNA / RNA separation buffer, and is compatible with the efficient separation and purification process of DNA and RNA silica gel.

[0102] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A method for separating, extracting and purifying nucleic acids from different cell components, characterized in that: A kit is used to separate, extract and purify nucleic acids from different cell components. The kit includes RNA binding solution, anhydrous ethanol, RNA rinsing solution 1 solution, RNA rinsing solution 2 solution, nuclear and cytoplasmic DNA / RNA separation buffer, chloroform, and DNA rinsing solution; the RNA binding solution is composed of 1-8M guanidine thiocyanate and 1M Tris-HCl, and the pH value is 7.4; the RNA rinsing solution 1 solution is composed of 80% ethanol and 2M guanidine hydrochloride; the RNA rinsing solution 2 solution is an 80% ethanol solution; the nuclear and cytoplasmic DNA / RNA separation buffer is composed of a lysis solution and a TRIZOL reagent in a volume ratio of 1:2, and the lysis solution is composed of 4M guanidine thiocyanate and 1M Tris-HCl, and the pH value is 7.4; The DNA washing solution is composed of 80% ethanol and 100 mM NaCl; comprising the following steps: S1. Separate cytoplasmic components, nuclear free components and chromosome components from cells; S2. Extraction of RNA from cytoplasmic fractions or nuclear free fractions: S21, adding RNA binding solution to the cytoplasmic component or the nuclear free component, and then adding anhydrous ethanol to mix; the volume ratio of the cytoplasmic component or the nuclear free component, the RNA binding solution and the anhydrous ethanol is 1:1:2-4; S22, incubating the mixed solution of S21 with the silica gel column to allow the RNA to bind to the silica gel column; S23, adding RNA rinsing solution 1 solution to the silica gel column in S22 for rinsing; S24, adding RNA rinsing solution 2 to the silica gel column in S23 for rinsing, and then spinning and eluting with pure water to obtain an RNA sample; S3. Co-extraction of chromatin-associated RNA and genomic DNA: S31, dissolve the chromosome components with nuclear DNA / RNA separation buffer, then add chloroform, centrifuge to mix and separate the layers; S32, taking the supernatant in S31 and incubating it with a silica gel column to allow the DNA to bind to the silica gel column; S33, adding DNA rinsing solution to the silica gel column in S32 for rinsing, and then spinning and eluting with pure water to obtain a genomic DNA sample; S34. Collect the flow-through of S32, add anhydrous ethanol and incubate it with a new silica gel column. The volume ratio of the flow-through to anhydrous ethanol is 1:1-2, so that the RNA is bound to the silica gel column. Then, process according to S23 and S24 to obtain a chromatin RNA sample.

2. A method for separating, extracting and purifying nucleic acids from different cell components according to claim 1, characterized in that: In S21, the RNA binding solution added to the cytoplasmic fraction consists of 5M guanidine thiocyanate and 1M Tris-HCl, pH=7.4; the RNA binding solution added to the nuclear free fraction consists of 6-7M guanidine thiocyanate and 1M Tris-HCl, pH=7.

4.

3. A method for separating, extracting and purifying nucleic acids from different cell components according to claim 1, characterized in that: In S31, the volume ratio of nuclear DNA / RNA separation buffer to chloroform is 4-6:

1.

4. A method for separating, extracting and purifying nucleic acids from different cell components according to claim 1, characterized in that: S1 separates cytoplasmic components, nuclear free components and chromosome components from cells, specifically comprising the following steps: S11, wash the cells with ice-cold PBS / 1 mM EDTA buffer and collect the cell pellet by centrifugation; S12, add ice-cold lysis buffer 1 to the cell pellet of S11, incubate on ice, gently pipette to mix the lysis solution, then cover it on an ice-cold sucrose cushion, centrifuge and collect the supernatant as a cytoplasmic component for standby use, and the remaining cell nuclear pellet proceeds to the next step; the lysis buffer 1 is composed of 10 mM Tris-HCl, 0.05% IGEPAL CA-630, 150 mM NaCl, 2 μL RNase inhibitor is added per 1 mL, pH=7.5; the sucrose cushion is prepared by dissolving nuclease-free sucrose in lysis buffer 1 at a mass percentage of 20-30%; S13. Add ice-cold PBS / 1 mM EDTA buffer to the cell nuclear pellet obtained in S12, remove PBS / EDTA without disturbing the pellet, resuspend the cell nuclear pellet in pre-cooled glycerol buffer, mix well, add an equal volume of ice-cold nuclear lysis buffer, mix well and incubate, then centrifuge to collect the supernatant as the nuclear free component for stand-alone use, and rinse the pellet with ice-cold PBS / 1 mM EDTA buffer, and collect the pellet as the chromosome-related component; the glycerol buffer consists of 20 mM Tris-HCl, 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, 0.125 mM PMSF, 50% glycerol, pH=7.9; the nuclear lysis buffer consists of 10 mM HEPES, 1 mM DTT, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M urea, 1% IGEPAL CA-630, pH=7.

6.

5. A method for separating, extracting and purifying nucleic acids from different cell components according to claim 4, characterized in that: In S11, every 5×10 6 -1×10 7 Add 1 mL of ice-cold PBS / 1 mM EDTA buffer to each cell.

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