Cell dissolution method

Abstract

To develop a cell dissolution method that has sufficiently high dissolution efficiency but does not inhibit subsequent enzymatic reactions.SOLUTION: A cell dissolution method includes the following steps: (a) adding a modifying surfactant to a cell to dissolve the cell; and then (b) adding an unmodifying surfactant to a cell lysate obtained in the step (a).SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for lysing cells. Specifically, the present invention relates to a method for lysing cells for preparing a sample suitable for single-cell gene expression analysis.

Background Art

[0002] To date, techniques for analyzing gene expression information, genomic sequence information, epigenomic information, etc. at the single-cell level have been developed and applied to elucidate various life phenomena, medical research, search for diagnostic markers, and search for therapeutic targets. Cells function by interacting with other cells and the extracellular matrix in organs and tissues. However, in most single-cell analysis techniques, at the stage of collecting cells, it is necessary to destroy organs and tissues and isolate fresh cells, resulting in the loss of histological information of the cells.

[0003] To avoid this problem, in recent years, techniques for performing gene analysis and the like without impairing the information on tissue sections have been developed. Most of the recently developed techniques employ an approach of directly analyzing the nucleic acids contained in tissue sections without isolating cells from the tissue sections. This approach can analyze the entire section, while being characterized by detecting nucleic acids without lysing the cells in the section (if the cells are lysed, the nucleic acids of different cells will be mixed). Due to this detection principle, there are problems such as low detection sensitivity and the detection target being limited to a part of the nucleic acid polymer (ends or hybridization targets of probe nucleic acids).

[0004] On the other hand, there is also an approach of physically cutting out cells from tissue sections with a laser. Its advantage is that since cells are physically isolated, in principle, it is possible to completely solubilize the nucleic acids to be analyzed and make them the analysis target.

[0005] Existing cell lysis methods for performing gene expression analysis on cells or tissue fragments (region of interest: ROI) isolated from tissue sections are roughly classified as follows (Non-Patent Documents 1 to 3). (i) Cells isolated from tissue sections, such as frozen tissue sections, or ROIs are lysed with a non-denaturing surfactant. Non-denaturing surfactants have the advantage of allowing cell lysis and enzymatic reactions to be performed consecutively in a single collection tube, as they do not inhibit the series of enzymatic reactions necessary for gene expression analysis (such as reverse transcription for synthesizing cDNA from mRNA and polymerase chain reaction (PCR) for DNA amplification). This method is commonly used when analyzing fresh single cells. On the other hand, tissue sections are usually fixed, dried, and stained with alcohol for morphological observation, and it has been reported that non-denaturing surfactants are insufficiently effective in dissolving them. (ii) Cells isolated from tissue sections such as frozen tissue sections or ROIs are lysed under denaturing conditions (such as guanidine isothiocyanate) or with proteinase. The advantage of this method is that cells can be sufficiently lysed, but because enzymatic reactions are inhibited, it is difficult to complete cell lysis and enzymatic reactions in a single collection tube, and for highly efficient analysis, it is desirable to purify nucleic acids after cell lysis. Nucleic acid purification involves many steps such as centrifugation and collection tube replacement, which is time-consuming and is particularly problematic when analyzing many single cells at once. Sample loss associated with purification is also a potential problem. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Chen, J., Suo, S., Tam, PP, Han, JJ, Peng, G. and Jing, N. (2017) Spatial transcriptomic analysis of cryosectioned tissue samples with Geo-seq. Nat Protoc, 12, 566-580. [Non-Patent Document 2] Nichterwitz, S., Chen, G., Aguila Benitez, J., Yilmaz, M., Strovall, H., Cao, M., Sandberg, R., Deng, Q. and Hedlund, E. (2016) Laser capture microscopy coupled with Smart-seq2 for precise apatial transcriptomic profiling. Nat Commun, 7, 121139. [Non-Patent Document 3] Singh, S., Wang, L., Schaff, DL, Sutcliffe, MD, Koeppel, AF, Kim, J., Onengut-Gumuscu, S., Park, KS, Zong, H. and Janes, KA (2019) In situ 10-cell RNA sequencing in tissue and biopsy tumor sample. Sci Rep, 9, 4836. [Overview of the project] [Problems that the invention aims to solve]

[0007] In techniques that use cell lysates as samples for subsequent enzymatic reactions, such as single-cell analysis, cell lysis is a problem. There was a need to develop a cell lysis method that was sufficiently efficient without inhibiting subsequent enzymatic reactions. [Means for solving the problem]

[0008] The inventors of the present invention have diligently conducted research to solve the above problems and have found that by isolating single cells from frozen sections using laser capture microdissection (hereinafter referred to as LCM) and quenching them with a non-denaturing surfactant (hereinafter referred to as non-denaturing surfactant) after using a denaturing surfactant (hereinafter referred to as denaturing surfactant), the inhibition of subsequent enzymatic reactions can be suppressed, thus completing the present invention.

[0009] In other words, the present invention provides the following: (1) The following steps: (a) Add a denaturing surfactant to the cells to lyse them, and then (b) Add a non-denaturing surfactant to the cell lysate obtained in step (a). A cell lysis method, including the following. (2) The method according to (1), wherein the non-denaturing surfactant is added such that the concentration of the non-denaturing surfactant is five times or more the concentration of the denaturing surfactant. (3) The method according to (1) or (2), wherein a modified surfactant is added in step (a) to a concentration of 0.05% to 3%. (4) The method according to any one of (1) to (3), wherein the denaturing surfactant is selected from the group consisting of sodium deoxycholate, sodium cholate, sodium dodecyl sulfate (SDS), and sodium lauroyl sarcosinate, and the non-denaturing surfactant is selected from the group consisting of Triton X-100, Nonidet P-40, Tween 20, and Brij 35. (5) The method described in any of (1) to (4), wherein step (a) is carried out in the presence of a protease. (6) The method according to any one of (1) to (5), wherein protein is added in step (b). (7) The method described in any of (1) to (6) above, wherein the cells are derived from tissue sections. (8) A method for preparing a sample for single-cell analysis, as described in any of (1) to (7). (9) A method for preparing a sample for gene expression analysis, as described in any of (1) to (7). (10) A cell lysis kit comprising a denaturing surfactant and a non-denaturing surfactant in separate containers, wherein the denaturing surfactant is added to the cells and the cells are lysed, and then the non-denaturing surfactant is added to the cell lysate. (11) The kit according to (10), wherein the non-denaturing surfactant is added such that the concentration of the non-denaturing surfactant is five times or more the concentration of the denaturing surfactant. (12) The kit according to (10) or (11), wherein a modified surfactant is added to a concentration of 0.05% to 3%. (13) A kit according to any one of (10) to (12), wherein the denaturing surfactant is selected from the group consisting of sodium deoxycholate, sodium cholate, sodium dodecyl sulfate (SDS), and sodium lauroyl sarcosinate, and the non-denaturing surfactant is selected from the group consisting of Triton X-100, Nonidet P-40, Tween 20, and Brij 35. (14) A kit according to any one of (10) to (13), further comprising a protease added together with a denaturing surfactant. (15) A kit according to any one of (10) to (14), further comprising a protein to be added together with a non-denaturing surfactant. (16) A kit in which the cells are derived from tissue sections, as described in any of (10) to (15). (17) A kit for preparing a sample for single-cell analysis, as described in any of (10) to (16). (18) A kit for preparing samples for gene expression analysis, as described in any of (10) to (16). A single-cell analysis method comprising analyzing a sample prepared using the method described in any of (1) to (9) or the kit described in any of (10) to (18). A method for analyzing gene expression, comprising analyzing a sample prepared using the method described in any of (1) to (9) or the kit described in any of (10) to (18). (21) The method according to (19) or (20), wherein the prepared sample is not purified of nucleic acids. [Effects of the Invention]

[0010] The present invention provides an efficient method for cell lysis. The cell lysate obtained by the method of the present invention does not inhibit subsequent enzymatic reactions (e.g., reverse transcription or polymerase chain reaction (PCR)). The cell lysate obtained by the present invention can be directly applied to cDNA synthesis and amplification without purifying RNA, and the amplified cDNA can be applied to gene expression analysis (RNA-seq) using next-generation sequencers. According to the present invention, cell lysis and enzymatic reactions can be carried out in a single reaction vessel.

[0011] In other words, according to the present invention, it is possible to achieve both sufficient cell lysis under denaturing conditions and completion of the subsequent enzymatic reaction without purification. In this respect, the present invention has features that fundamentally differ from the prior art.

[0012] By using the present invention, it is possible to obtain a number of detectable genes and gene expression patterns comparable to those obtained from fresh cells isolated while still alive, even from dead cells such as cells in frozen tissue sections, enabling efficient and highly accurate gene expression analysis. Furthermore, the present invention makes it possible to perform mRNA splice variant analysis, which was not previously possible for single cells in tissue sections. [Brief explanation of the drawing]

[0013] [Figure 1]Figure 1 shows the results of single-cell gene expression analysis of frozen cell blocks of mouse ES cells. Dissociated fresh cells in Figure 1A are the results of analyzing gene expression by real-time PCR after lysing fresh single ES cells collected from a cell suspension with a denaturing surfactant and a non-denaturing surfactant according to the present invention. LCM (Triton X-100) in Figure 1A is the result of analyzing gene expression by real-time PCR after treating single cells collected from a frozen cell block of mouse ES cells with only Triton X-100. LCM (DRaqL) in Figure 1A is the result of analyzing gene expression by real-time PCR after lysing single cells collected from a frozen cell block of mouse ES cells with a denaturing surfactant and a non-denaturing surfactant according to the present invention. Figure 1B shows the results of RNA-seq. Sample from fresh single ES cells vs. sample from fresh single ES cells (left), sample from fresh single ES cells vs. sample obtained by the method of the present invention (middle), and sample obtained by the method of the present invention vs. sample obtained by the method of the present invention (right). [Figure 2] Figure 2 is a photograph of a portion containing oocytes and granulosa cells in a mouse ovarian tissue section (cresyl violet staining). The black scale bar is 50 μm. The circles in the photograph are the cells collected by LCM. [[ID=​​​Figure 4 shows the results of splice variant analysis that retained histological information of oocytes in mouse ovarian follicles. The upper panel is a diagram showing the exon connection patterns detected in the Cenpa gene in oocytes. The lower panel is a diagram showing the exon-intron structure of the Cenpa gene in the mouse genome. [Figure 5] Figure 5 is a graph showing the examination results of the expression level (Ct value) of the Arpb gene when cell lysis was performed using various combinations of denaturing surfactants and non-denaturing surfactants. In each combination, the three white bars on the left are the results when no non-denaturing surfactant was used, and the three gray bars on the right are the results when a denaturing surfactant and a non-denaturing surfactant were used in combination. [Figure 6] Figure 6 is a graph showing the results of examining the influence of the presence or absence of surfactants on gene expression analysis. Cell lysis (SDCs) + Quenching (Triton X-100) is the result when cells were lysed using both a denaturing surfactant and a non-denaturing surfactant, Cell lysis (Triton X-100) + Quenching (H2O) is the result when cells were lysed using only a non-denaturing surfactant, and Quenching- (H2O + H2O) is the result when cells were collected in water. In this experiment, the expression level (Ct value) of the Arpb gene was measured by real-time PCR. [Figure 7] Figure 7 is a graph showing the results of examining the influence of the presence or absence of protease treatment on gene expression analysis. DRaqL-Protease-Smart-seq2 is the result when treated with a denaturing surfactant containing protease and then with a non-denaturing surfactant, Protease-Smart-seq2 is the result when protease treatment was performed without deoxycholic acid, and Smart-seq2 is the result when solubilization was performed with only TritonX-100. In this experiment, the expression level (Ct value) of the Arpb gene was measured by real-time PCR. The P value obtained by performing a t-test on the difference in the average Ct values between DRaqL-Protease-Smart-seq2 and Protease-Smart-seq2 is also shown. [Modes for carrying out the invention]

[0014] In one embodiment, the present invention provides the following method: The following steps: (a) Add a denaturing surfactant to the cells to lyse them, and then (b) Add a non-denaturing surfactant to the cell lysate obtained in step (a). A cell lysis method including the following.

[0015] The cells may be any type of cell, and are not particularly limited. The cells may be animal cells, plant cells, or microbial cells. Preferably, the cells are mammalian cells. The cells may be dead cells or living cells. Examples of dead cells include, but are not limited to, cells in tissue specimens, such as cells in frozen tissue sections. Live sperm or spores may also be lysed using the method of the present invention.

[0016] For example, single target cells may be recovered from frozen tissue sections using laser capture microdissection (LCM), and then lysed using the method of the present invention.

[0017] Tissue fragments (ROIs) may be used as cells in the method of the present invention. Tissue fragments may be obtained from dead tissue such as tissue specimens, or from living tissue such as biopsies. In this specification, single cells, multiple cells (e.g., cell populations, cell aggregates), tissue fragments, etc., are collectively referred to as cells.

[0018] Denaturing surfactants are surfactants that strongly solubilize proteins by denaturing them and destroying their higher-order structure. Enzymes are usually inactivated in the presence of denaturing surfactants. Denaturing surfactants are known to those skilled in the art. In the present invention, anionic surfactants and amphoteric surfactants are preferably used as denaturing surfactants. Examples of denaturing surfactants that can be used in the method of the present invention include sodium deoxycholate, other deoxycholates, sodium dodecyl sulfate (SDS), other dodecyl sulfate esters, cholates, hexadecyl sulfate, decyl sulfate, dodecylbenzenesulfonate, 1-tetradecanesulfonate, cetylsulfonate, dimethyl 5-sulfisophthalate, 1-pentadecanesulfonate, 1-octadecanesulfonate, dodecylbenzenesulfonate, laurylsulfonate, 1-decanesulfonate, alkylnaphthalenesulfonate, monododecyl phosphate, polyoxyethylene lauryl ether acetate, α-olefin sulfonate. Examples of modified surfactants that are preferably used in the present invention include, but are not limited to, oleates, N-oleoyl sarcosine salts, palmitates, stearates, lauroyl sarcosine salts, N-decanoyl sarcosine salts, myristicates, laurates, CHAPS, CHAPSO, tetradecyldimethyl(3-sulfopropyl)ammonium hydroxide intramolecular salt, hexadecyldimethyl(3-sulfopropyl)ammonium hydroxide intramolecular salt, decyldimethyl(3-sulfopropyl)ammonium hydroxide intramolecular salt, dodecyldimethyl(3-sulfopropyl)ammonium hydroxide intramolecular salt, and dimethyl(n-octyl)(3-sulfopropyl)ammonium hydroxide intramolecular salt.

[0019] In step (a), a denaturing surfactant is added at a concentration sufficient to lyse cells and to eliminate its denaturing effect (quench) in step (b). The concentration of the denaturing surfactant used in step (a) can be determined by a person skilled in the art, taking into consideration the type of cells; the type of denaturing surfactant; cell lysis conditions such as temperature, pH, salt concentration, and buffer type; the type and concentration of the non-denaturing surfactant used in the next step; and the type of enzyme used in subsequent reactions (e.g., mRNA expression analysis). Generally, a concentration range of 0.05% to 3% is exemplified for the denaturing surfactant, but concentrations below or above this range may also be used. Typical examples include, but are not limited to, 0.2% to 2% deoxycholic acid or 0.05% to 0.2% SDS when mRNA expression analysis is performed in a subsequent step.

[0020] Quenching a denaturing surfactant means eliminating its protein-denaturing effect, preventing it from inhibiting enzymes used in subsequent analyses, and ensuring that the enzymes used in subsequent analyses maintain a level of activity sufficient to perform the analysis.

[0021] An example of a method for determining the upper limit of the concentration of the denaturing surfactant used in step (a) is shown below (when gene expression analysis is performed as a subsequent analysis). This method includes steps (i) to (iii). (i): Prepare multiple nucleic acid samples (e.g., RNA equivalent to that contained in a single mammalian cell) dispensed from the same nucleic acid sample solution, and use these as starting samples. Perform steps (a) and (b) for each sample. However, in order to explore the upper limit of the denaturing surfactant used in step (a), try two or more different concentrations. (ii): For nucleic acid samples dispensed from the same nucleic acid sample solution as in step (i), perform only step (b). (iii) Analyze the products of process (i) and process (ii) (e.g., cDNA amplification, gene expression analysis), compare the data obtained from both, and determine the highest concentration of denaturing surfactant in process (a) that is below a predetermined standard (e.g., less than 2 times) when the difference in nucleic acids detected in process (i) and process (ii) (e.g., expression level of a specific gene) is less than 2 times, and set this as the upper limit for use in actual analysis.

[0022] Step (a) may be carried out while heating. The heating temperature can be appropriately determined by a person skilled in the art, taking into consideration the type of modified surfactant, etc. The heating temperature is usually 50°C to 80°C, preferably 65°C to 75°C, but is not limited to these temperatures.

[0023] Step (a) may be carried out in the presence of a protease. Doing so can increase the efficiency of subsequent analytical steps such as gene expression analysis and enable stable analysis. The protease used in this step is preferably one that has sufficient activity in the presence of a denaturing surfactant. Examples include, but are not limited to, Qiagen protease (serine protease derived from Bacillus bacteria), proteinase K (Tritirachium album), proteinase from Aspergillus melleus (also known as protease M [Amano]), subtilisin, and other subtilisin-like proteases. By carrying out step (a) in the presence of a protease, the lysis efficiency of cells in tissues fixed using cross-linking fixatives such as formalin or glutaraldehyde can be further improved.

[0024] The product obtained in step (a) is a lysis solution of cell or tissue fragments (ROI) containing a denaturing surfactant, which can be described as a lysis solution in which the denaturing effect of the denaturing surfactant can be eliminated by the addition of a non-denaturing surfactant.

[0025] In the method of the present invention, a non-denaturing surfactant is added (step (b)) to eliminate (quench) the denaturing effect of the denaturing surfactant during cell lysis obtained in step (a) using a denaturing surfactant.

[0026] Nondenaturing surfactants are surfactants that do not denature water-soluble proteins, have a weak denature effect, or have a protective effect on water-soluble proteins. Nondenaturing surfactants are known to those skilled in the art. In the present invention, nonionic surfactants are preferably used as nondenaturing surfactants. Examples of nondenaturing surfactants that can be used in the method of the present invention include Triton X-100, Triton X-114, Triton X-305, Triton X-405, Nonidet P-40, Tween 20, Tween 40, Tween 80, polyoxyethylene (23) lauryl ether (Brij 35), polyoxyethylene (20) cetyl ether (Brij 58), octyl glucoside, n-octyl-α-glucopyranoside, n-octyl-β-glucopyranoside, n-octyl-D17-β-D-glucopyranoside, n-octyl- β-D-galactopyranoside, n-octyl-β-D-galactopyranoside-d7, n-octyl-β-D-maltopyranoside, octylmaltoside, octyl-β-D-selenoglucoside, n-octyl-β-D-thioglucopyranoside, n-octyl-β-D-thiomaltopyranoside, N-dodecyl-β-D-maltopyranoside, dodecyl-β-D-selenomaltoside, Anameg-7, Anapoe-20, Anapoe-3 5, Anapoe-58, Anapoe-80, Anapoe-C10E6, Anapoe-C10E9, Anapoe-C12E8, Anapoe-C12E9, Anapoe-C12E10, Anapoe-C1 3E8, Anapoe-NID-P40, C-HEGA-8, C-HEGA-9, C-HEGA-10, C-HEGA-11, HEGA-8, HEGA-9, HEGA-10, HEGA-11, Chobimalt, C yclofos-7, CYGLU-3, CYGLU-4, CYMAL-1, CYMAL-2, CYMAL-3, CYMAL-4, CYMAL-5, CYMAL-6, CYMAL-7, CYMAL-6 neopentyl glycol, CYMAL-7 neopentyl glycol, n-decyl-β-D-glucopyranoside, decyl glucose, n-decyl-α-D-maltopyranoside, n-decyl-β-D-maltopyranoside, decyl maltose,Decyl-β-D-selenomaltoside, n-decyl-β-D-thioglucopyranoside, decyl glucose neopentyl glycol, decyl maltose neopentyl glycol, 2,6-dimethyl-4-heptyl-β-D-maltoside, n-dodecyl-α-D-glucopyranoside, n-dodecyl-β-D-glucopyranoside, n-dodecyl-d25-β-D-maltopyranoside, n-dodecyl-β-D-thiomaltopyranoside, n-heptyl-β-D-glucopyranoside, heptyl-β-D-selenoglucoside, n-heptyl-β-D-thioglucopyranoside, n- Hexadecyl-β-D-maltopyranoside, hexaethylene glycol monodecyl ether, IPTG, decyl maltose neopentyl glycol, lauryl maltose neopentyl glycol, octyl glucose neopentyl glycol, Mega-8, Mega-9, Mega-10, monoolein, monopalmitolein, n-nonyl-β-D-glucopyranoside, n-nonyl-β-D-maltopyranoside, n-nonyl-β-D-thioglucopyranoside, octaethylene glycol monododecyl ether, pentaethylene glycol monodecyl ether, Pluronic F-68, Pluronic F-127, 2-Propyl-1-Pentyl Maltopyranoside, L-(+)-Selenomethionine, 12-Selenotetraethylene glycol monooctyl ether, Sucrose monododecanoate, n-Tetradecyl-β-D-Maltopyranoside, Tetraethylene glycol monooctyl ether, Tandem Facial Amphiphile (TFA), n-tridecyl-β-D-maltopyranoside, Cy-Tripglu, Ph-Tripglu, n-undecyl-α-D-maltopyranoside, n-undecyl-β-D-maltopyranoside, undecyl-β-D-selenomaltoside, n-undecyl-β-D-thiomaltopyranoside, w-undecylenyl-β-D-maltopyranoside, n-hexadecyl-β-D-maltopyranoside, n-dodecyl-β-D-thiomaltopyranoside, CYMAL-5 neopentyl glycol, n-dodecyl-α-D-maltopyranoside, n-dodecyl-β-D-maltopyranoside, deoxy-BIGCHAP, hexaethylene glycol monooctyl ether, n-octyl-α-D-glucopyranoside,Examples of non-denaturing surfactants preferred in this invention include, but are not limited to, BisMalt-18, BisMalt-20, BisMalt-22, BisMalt-24, BisMalt-28, and digitonin. Examples of non-denaturing surfactants preferred in this invention include, but are not limited to, Triton X-100 (t-octylphenoxypolyethoxyethanol), Nonidet P-40 (octylphenoxypoly(ethyleneoxy)ethanol), Tween 20 (polyoxyethylene sorbitan monolaurate), and Brij 35 (polyoxyethylene(23) lauryl ether).

[0027] In step (b), it is preferable that the non-denaturing surfactant is added at a concentration that sufficiently quenches the denaturing effect of the denaturing surfactant and does not affect the subsequent enzymatic reaction. The concentration of the non-denaturing surfactant used in step (b) can be appropriately determined by a person skilled in the art, taking into consideration the type of cell; the type of non-denaturing surfactant; quenching conditions such as temperature, pH, salt concentration, and buffer type; and the type of enzyme used in the subsequent enzymatic reaction.

[0028] It is preferable to make the concentration of the non-denaturing surfactant added in step (b) higher than the concentration of the denaturing surfactant. For example, the concentration of the non-denaturing surfactant may be 5 times or more, 7 times or more, or 10 times or more than the concentration of the denaturing surfactant. Preferably, the concentration of the non-denaturing surfactant is 10 times or more than the concentration of the denaturing surfactant. For example, the concentration of the non-denatured surfactant may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 times, 10-20 times, 20-30 times, 30-40 times, 40-50 times, 50-60 times, 60-70 times, 70-80 times, 80-90 times, 90-100 times, 100-150 times, 150-200 times, 10-200 times, 10-100 times, 20-100 times, etc. A typical example is the use of 7% to 8% Triton X-100 (where 0.5% deoxycholic acid is used in step (a)), but this example is not limited to this case. The concentration ratio of the modified surfactant to the non-modified surfactant is the final concentration ratio in step (b).

[0029] Step (b) may be carried out after adding a protein. Proteins are known to act protectively against enzymes and are thought to help eliminate the denaturing effect of the denaturing surfactant. The type and concentration of the protein can be appropriately determined considering the type and concentration of the denaturing surfactant, the type of enzyme used in subsequent steps, etc. Examples of proteins to be added include, but are not limited to, bovine serum albumin, saprophytic fungal subtilisin protease, anti-ribonuclease antibodies, and RNase inhibitors. The concentration of the protein may be 0.01% or higher (concentration after addition), for example, 0.1%, 0.15%, 0.2%, 0.5%, etc. A typical example is 0.15% bovine serum albumin, but is not limited to this example.

[0030] Step (b) may be carried out while heating. The heating temperature can be appropriately determined by those skilled in the art, taking into consideration the type of non-denaturing surfactant, etc. The heating temperature is usually 50°C to 80°C, preferably 65°C to 75°C, but is not limited to these temperatures.

[0031] The product obtained in step (b) is a lysis solution for cell or tissue fragments (ROI) containing a denaturing surfactant and a non-denaturing surfactant, which can be described as a lysis solution in which subsequent enzymatic reactions are not inhibited. The product obtained in step (b) can be used directly, for example, for gene expression analysis. The product obtained in step (b) may also be used as a sample for single-cell analysis.

[0032] Preferred combinations of denatured and undenatured surfactants used in the method of the present invention include, but are not limited to, sodium deoxycholate and Triton X-100, sodium deoxycholate and Tween 20, sodium deoxycholate and Brij 35, sodium N-lauroyl sarcosinate and Tween 20, sodium N-lauroyl sarcosinate and Triton X-100, sodium N-lauroyl sarcosinate and Brij 35, sodium cholate and Tween 20, sodium cholate and Triton X-100, sodium cholate and Brij 35, SDS and Triton X-100, and SDS and Tween 20.

[0033] When RNA is the target of analysis in a subsequent step of the cell lysis method of the present invention (for example, when performing gene expression analysis), the following points should be noted. Since RNA is more susceptible to hydrolysis under alkaline conditions, it is preferable to maintain a neutral or acidic pH when applying denaturing surfactants. To avoid hydrolysis of RNA, it is preferable to keep the temperature below 80°C when heating is applied to the surfactant. If subsequent steps involve reverse transcription of RNA, it is preferable to thermally denaturate the RNA after quenching. Furthermore, it is preferable to add the reverse transcription substrate (deoxynucleotide triphosphate) and primer simultaneously with the denaturing surfactant. It is preferable to add an RNase inhibitor along with a denaturing surfactant to suppress the enzymatic degradation of RNA.

[0034] In this specification, adding a surfactant to cells means bringing the surfactant into contact with the cells, and includes adding a surfactant to a sample containing cells, and adding a sample containing cells to a solution containing a surfactant.

[0035] In another embodiment, the present invention provides the following kit: A cell lysis kit comprising a denaturing surfactant and a non-denaturing surfactant in separate containers, wherein the denaturing surfactant is added to the cells and the cells are lysed, and then the non-denaturing surfactant is added to the cell lysate.

[0036] The kit of the present invention is used to carry out the cell lysis method of the present invention as described above. The kit is usually accompanied by instructions for use.

[0037] The kit of the present invention includes one container containing a denatured surfactant and another container containing a non-denatured surfactant. The form of the denatured and non-denatured surfactants in the containers is not particularly limited; they may be solids such as powders or solutions. The amount of the denatured and non-denatured surfactants in the containers can also be appropriately determined considering the type and concentration of the surfactants used, the type of cells, the amount of cells to be treated, the number of treatments, etc. The material and shape of the containers are also not particularly limited; any material and shape suitable for storing surfactants is acceptable.

[0038] The kit of the present invention may further include a protein to be added together with a non-denaturing surfactant. The protein may be in a separate container or in a container together with the non-denaturing surfactant.

[0039] The cells in the cell lysate obtained using the method and kit of the present invention are sufficiently lysed because they have been treated with a denaturing surfactant. Furthermore, since the denaturing effect of the surfactant is quenched in the cell lysate obtained using the method and kit of the present invention, it is very advantageous when performing subsequent enzymatic reactions. The cell lysate obtained using the method and kit of the present invention may also be used as a sample for single-cell analysis. The cell lysate obtained using the method and kit of the present invention is suitable as a sample for, for example, gene expression analysis, genome sequence information analysis, epigenomic information analysis, etc.

[0040] A further advantage of the present invention is that, when a subsequent enzymatic reaction is required, cell lysis and the enzymatic reaction can be carried out in a single reaction vessel. In such cases, the need to purify nucleic acids after cell lysis can be eliminated, thus eliminating sample loss associated with purification. By using the lysis method of the present invention, for example, when performing single-cell analysis, a large number of single cells can be analyzed simultaneously and rapidly, which is efficient.

[0041] Therefore, the present invention provides the following method. A single-cell analysis method comprising analyzing a sample prepared using the method or kit of the present invention, and A method for analyzing gene expression, comprising analyzing a sample prepared using the method or kit of the present invention.

[0042] Unless otherwise specified, terms used herein have the meanings commonly understood in the fields of biology, biochemistry, chemistry, genetics, molecular biology, medicine, and pharmacology.

[0043] The present invention will be described in more detail and specifically below with reference to examples, but these examples are not intended to limit the scope of the present invention. [Examples]

[0044] Example 1: Single-cell gene expression analysis of frozen mouse ES cell blocks Experimental method Mouse ES cells cultured under "2iLIF" conditions, known to exhibit homogeneous gene expression, were suspended in trypsin, pelletized, flash-frozen in liquid nitrogen, sectioned, fixed with alcohol, stained with cresyl violet, and individual cells were collected by LCM. 0.5% sodium deoxycholate (w / v) was added to the collected cells, and 7.6% Triton X-100 (v / v, final concentration) was added to the resulting cell lysate. Specifically, cell lysis was performed in 6.4 μL of 0.5% deoxycholic acid aqueous solution (step (a)), and 2.8 μL of 25% Triton X-100 aqueous solution was added in the quenching step (step (b)). The final concentration of deoxycholic acid in step (b) was 0.348%, the final concentration of Triton X-100 was 7.6%, and the concentration ratio was approximately 22-fold. Gene expression analysis of the obtained samples was performed using real-time PCR and RNA-seq without nucleic acid purification. For comparison, fresh mouse ES cells isolated from a suspension were treated in the same manner as described above, and gene expression analysis was performed. To compare the solubility conditions with those of a non-denaturing surfactant, single cells isolated from a frozen block of mouse ES cells were solubilized with Triton X-100 alone, and gene expression analysis was performed.

[0045] Experimental results Real-time PCR analysis showed that, compared to solubilization with Triton X-100 alone, solubilization using the method of the present invention significantly improved the efficiency of gene expression detection, confirming that gene expression analysis could be performed with the same efficiency as with fresh cells (Figure 1A). RNA-seq analysis also showed that solubilization using the method of the present invention yielded the same number of detectable genes and gene expression patterns as with fresh cells (Figure 1B). These results confirm that the present invention enables highly accurate gene expression analysis of cells recovered from frozen sections. [Examples]

[0046] Example 2: Single-cell gene expression analysis of oocytes and granulosa cells in mouse ovarian follicles while preserving histological information. Experimental method After excising mouse ovaries, they were flash-frozen in liquid nitrogen, sectioned, fixed with alcohol, stained with cresyl violet, and individual cells were collected using LCM. Cells adjacent to and not adjacent to oocytes were collected (Figure 2). 0.5% sodium deoxycholate (w / v) was added to the collected cells, and 7.6% Triton X-100 (v / v, final concentration) was added to the resulting cell lysates. Gene expression analysis of the obtained samples was performed using real-time PCR and RNA-seq.

[0047] Experimental results We detected differences in gene expression caused by histological information of granulosa cells (whether they were adjacent to oocytes or not) (Figure 3). Furthermore, the number of genes detected in oocytes was equivalent to the gene expression data for raw single oocytes reported in previous studies. These results demonstrate that the present invention enables gene expression analysis that retains histological information in actual tissues. [Examples]

[0048] Example 3: Splice variant analysis of oocytes in mouse ovarian follicles while preserving histological information. Experimental method After removing mouse ovaries, individual oocytes were collected using the same method as in Example 2. 0.5% sodium deoxycholate (w / v) was added, and 7.6% Triton X-100 (v / v, final concentration) was added to the resulting cell lysates. The exon junctions of mRNA in the obtained samples were analyzed using paired-endRNA-seq.

[0049] Experimental results Multiple exon junctions were detected in the genes whose expression was detected (Figure 4, upper panel). In particular, a splice variant (Cenpa splice variant) that is considered to play an important role in oocyte growth was detected (Figure 4, lower panel). These data demonstrate that the present invention enables single-cell splice variant analysis from frozen sections.

[0050] Example 4: Cell lysis and gene expression analysis using various combinations of denaturing and non-denaturing surfactants Experimental method To compare the quench efficiency of combinations of denaturing and non-denaturing surfactants, cDNA amplification was performed from 10 pg of ES cell-derived RNA. The denaturing surfactants used were 0.156% sodium deoxycholate (SDc), 0.156% sodium cholate, and 0.078% N-lauroylsarcosine sodium salt. The non-denaturing surfactants (final concentration 7.6%) used were TritonX-100, Brij L23 (lauryl ether (Brij35)), and Tween20. The expression levels of the Arbp gene were examined in samples obtained using the above surfactant combinations (see Figure 5) by real-time PCR.

[0051] Experimental results The results are shown in Figure 5. It was found that, regardless of whether the combination was a denaturing surfactant or a non-denaturing surfactant, the denaturing surfactant could be quenched by the non-denaturing surfactant with almost the same efficiency as the combination of SDc and TritonX-100, enabling successful gene expression analysis.

[0052] Example 5: Effect of the presence or absence of surfactants on gene expression analysis Experimental method Mouse ES cells cultured under 2iLIF conditions were suspended in trypsin, pelletized, flash-frozen in liquid nitrogen, sectioned, fixed with alcohol, stained with cresyl violet, and individual cells were collected by LCM. Collected cells were treated with 0.5% sodium deoxycholate (w / v), and the resulting cell lysates were treated with 7.6% Triton X-100 (v / v, final concentration) (Cell lysis (SDCs) + Quenching (Triton X-100)). Real-time PCR was performed on the obtained samples to analyze the expression level of the Arbp gene. As a comparative experiment, the expression levels of the Arbp gene were analyzed in the same manner as above for two groups: one group in which cells were collected in water (Quenching- (H2O + H2O)) and another group in which cells were lysed with Triton X-100 alone (Cell lysis (Triton X-100) + Quenching (H2O)).

[0053] Experimental results The results are shown in Figure 6. Compared to two comparison groups (Quenching- (H2O + H2O) and Cell lysis (Triton X-100) + Quenching (H2O)), the group using the method of the present invention (Cell lysis (SDCs) + Quenching (Triton X-100)) showed a difference of more than 3 cycles in the average Ct value of the average real-time PCR (more than 9 times in terms of cDNA amount). These results confirm that the elution efficiency was significantly improved and the amount of cDNA was significantly increased in the group lysed according to the present invention.

[0054] Example 6: Combination of cell lysis and protease treatment according to the present invention Experimental method Frozen mouse ovarian sections were fixed in formalin (10% formalin, room temperature, 24 hours). They were then stained with cresyl violet. Single granulosa cells were collected using LCM in a 0.3% sodium deoxycholate solution containing 288 mAU of heat-unstable protease (Qiagen protease). The protease reaction was performed at 50°C for 10 minutes, followed by thermal inactivation of the protease at 80°C for 15 minutes. Deoxycholic acid was then quenched with 7.6% TritonX-100 (DRaqL-Protease-Smart-seq2), and cDNA amplification was performed using the Smart-seq2 method. As a control group, the same experiment was compared with protease treatment without deoxycholic acid (Protease-Smart-seq2) and with solubilization using TritonX-100 alone (Smart-seq2).

[0055] Experimental results The results are shown in Figure 7. When comparing the expression levels of the Arbp gene using real-time PCR, it was found that gene expression could be quantified more efficiently and stably in the group that underwent cell lysis combined with protease treatment (DRaqL-Protease-Smart-seq2) than in the other groups. [Industrial applicability]

[0056] The present invention allows for efficient lysis of cells. Therefore, it can improve the efficiency of analyses such as single-cell gene expression analysis. Consequently, the present invention is widely applicable to the development of pharmaceuticals and the elucidation of disease onset mechanisms.

Claims

1. The following steps: (a) adding a denaturing detergent to the cells to lyse the cells; (b) adding a non-denaturing detergent to the cell lysate obtained in step (a); A method for lysing cells, comprising:

2. 2. The method of claim 1, wherein the non-denaturing surfactant is added so that the concentration of the non-denaturing surfactant is at least five times the concentration of the denaturing surfactant.

3. 2. The method of claim 1, wherein the denaturing surfactant is added in step (a) to a concentration of 0.05% to 3%.

4. 2. The method of claim 1, wherein the denaturing surfactant is selected from the group consisting of sodium deoxycholate, sodium cholate, sodium dodecyl sulfate, and sodium lauroyl sarcosinate, and the non-denaturing surfactant is selected from the group consisting of t-octylphenoxypolyethoxyethanol, octylphenoxypoly(ethyleneoxy)ethanol, polyoxyethylene sorbitan monolaurate, and polyoxyethylene (23) lauryl ether.

5. 2. The method of claim 1, wherein step (a) is carried out in the presence of a protease.

6. The method of claim 1 , wherein a protein is added in step (b).

7. A kit for cell lysis comprising a denaturing detergent and a non-denaturing detergent in separate containers, wherein the denaturing detergent is added to cells to lyse the cells, and then the non-denaturing detergent is added to the cell lysate.

8. 8. The kit according to claim 7, wherein the non-denaturing surfactant is added so that its concentration is at least five times the concentration of the denaturing surfactant.

9. 8. The kit according to claim 7, wherein the denaturing surfactant is added to a concentration of 0.05% to 3%.

10. 8. The kit of claim 7, wherein the denaturing surfactant is selected from the group consisting of sodium deoxycholate, sodium cholate, sodium dodecyl sulfate, and sodium lauroyl sarcosinate, and the non-denaturing surfactant is selected from the group consisting of t-octylphenoxypolyethoxyethanol, octylphenoxypoly(ethyleneoxy)ethanol, polyoxyethylene sorbitan monolaurate, and polyoxyethylene(23) lauryl ether.

11. 8. The kit of claim 7, further comprising a protease added with the denaturing detergent.

12. 8. The kit of claim 7, further comprising a protein added with a non-denaturing detergent.

13. A single-cell analysis method, comprising analyzing a sample prepared using the method according to any one of claims 1 to 6 or the kit according to any one of claims 7 to 12.

14. A method for analyzing gene expression, comprising analyzing a sample prepared using the method according to any one of claims 1 to 6 or the kit according to any one of claims 7 to 12.

Images