JP2012125201A - Method for producing standard cell sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a cell sample which can simply be produced in a series of processes from a preliminary treatment process for extracting and purifying a nucleic acid to a process for detecting a nucleic acid amplification reaction and an amplified product, can be stored for a long period, and can be used for process accuracy control, in nucleic acid analysis such as gene expression amount inspection.SOLUTION: The method for producing the standard cell sample is characterized by having an inactivation treatment process comprising immersing a transformed cell to which an expression vector integrated with a standard nucleic acid whose base sequences are wholly or partially known is introduced, in an inactive liquid capable of holding the nucleic acid in the cell and capable of controlling the change of an expression profile, and the method for producing the standard cell sample is characterized in that an mRNA synthesized using the standard nucleic acid as a template exists in the transformed cell, and a protein originated from the standard nucleic acid is not expressed.

Description

  The present invention relates to a method for producing a standard cell sample that can be used for process accuracy control of nucleic acid analysis and the like.

  In recent years, it has been clarified that there is a multistage carcinogenesis mechanism until normal cells become cancerous (for example, see Non-Patent Documents 1 and 2). Specifically, accumulation of a plurality of gene abnormalities including a DNA repair gene, a tumor suppressor gene, and an oncogene is required for canceration of normal cells. In addition, activation of oncogenes and overexpression of growth factors are considered to be involved in cancer progression and malignant transformation. Thus, the development, growth, and metastasis of cancer involve the promotion or suppression of the expression of many genes. Thus, by measuring the expression levels of these genes, it is possible to estimate the presence of cancer at the gene level and to classify cancer types.

  If cancer can be detected and classified according to the degree of progression, differentiation, type, etc. quickly and easily, cancer can be detected at an early stage, and an appropriate treatment method can be selected for each patient. It is possible to make a suitable treatment policy. As a result, unnecessary treatment can be avoided, the burden on the patient can be minimized, and medical costs can be reduced. For this reason, there is a demand for the development of a method that can rapidly and accurately perform genetic analysis of oncogenes in specimens.

  In general, when analyzing the expression level of a human gene, RNA is extracted from a tissue or the like, and an amplification reaction of a nucleic acid derived from the gene to be analyzed is performed by RT (Reverse transcription) -PCR (Polymerase Chain Reaction). Since such analysis is performed through a plurality of processes, accurate results can be obtained only when each process is appropriately performed. For this reason, the accuracy control of each process is important.

  The accuracy control of the process is usually performed by processing a standard sample in the same process as the target sample and analyzing whether the obtained result is in the correct range. In the case of a plurality of steps, the accuracy control can be performed more easily by using a standard sample capable of performing the accuracy control for all the steps. For example, in the gene expression level test, in order to guarantee the entire process of nucleic acid extraction / purification from the target sample, which is the pretreatment process, and the nucleic acid amplification / detection process, it is always stable in all processes. It is preferable to use a positive standard sample.

  In conventional gene expression level tests, human-derived clinical samples (such as tissue fragments and blood cells) have been used as standard samples. Because clinical samples vary widely between samples due to individual differences, etc., in general, one clinical sample is subdivided and frozen in order to prevent deterioration. Used in the detection process. However, the expression level of genes varies depending on the site of the tissue, and the expression profile (or expression pattern) is not constant for each cell. For this reason, an appropriate clinical sample has to be used as a standard sample for each type of target nucleic acid to be analyzed. In addition, even if the sample is subdivided from one clinical sample, it is difficult to make each sample uniform, and as a result, the measurement result of the expression level of the standard sample varies and the reproducibility is poor. There is. Furthermore, because it is a frozen sample, it requires a process of thawing, mixing and liquefying before use, and adding reagents as appropriate to stabilize, and it cannot be immediately used for inspection as a standard sample. It was.

  A method of using an artificially prepared sample instead of a clinical sample as a standard sample for quality control is also disclosed. For example, a pseudo-tissue sample in which nucleic acid or cells are held in a polymer gel holder, or a pseudo-tissue sample in which at least a part of the surface of a polymer gel holder holding nucleic acids or cells is covered with a protector A method of using as a standard sample for accuracy control when performing gene analysis using a tissue excised from a living body as a specimen is disclosed (for example, see Patent Documents 1 and 2).

JP 2006-136310 A JP 2008-237109 A

Feelon E.E. R. (Fearon, ER) et al., Cell, 1990, 61, 759-767. Sugimura T. (Sugimura, T.), Science, 1992, 258, 603-607.

The pseudo-tissue sample described in Patent Documents 1 and 2 can be used as a standardized standard sample by using a nucleic acid prepared in advance uniformly. On the other hand, when cells are used, even if they are human-derived cells that have been cultivated, the expression profile of the gene is affected by the culture state (variation of culture operation, number of cell divisions). It is difficult to prepare a pseudo-tissue sample with a constant expression profile.
In addition, nucleic acids and cells are easily degraded and denatured. In particular, the expression profile of cells varies with time. For this reason, pseudo-tissue samples using nucleic acids and cells cannot be stably stored for a long period of time, and need to be prepared at the time of use in order to perform sufficient accuracy control.

  Furthermore, when a clinical sample to be analyzed does not have a firm connective tissue, such as a body fluid such as blood or feces, in order to use the pseudo tissue sample as a standard sample, first, a pseudo tissue sample The polymer gel holder and the protective body are dissolved by heating, mixed and homogenized, and then cooled to a temperature suitable for inspection. In addition, there is a problem that it takes time and effort to manufacture.

  The present invention can be easily produced in a series of steps from a pretreatment step of extracting and purifying a nucleic acid to a nucleic acid amplification reaction and an amplification product detection step in nucleic acid analysis such as gene expression level inspection. It is an object to provide a method for producing a cell sample that can be stored and can be used for process accuracy control.

  As a result of intensive studies to solve the above problems, the present inventors introduced an expression vector incorporating a nucleic acid that can be used as a control in a nucleic acid amplification step, and then immersed in an inactivation liquid to express an expression profile. Cells can be easily produced and stored stably for a long period of time, and the quality control of all steps from the nucleic acid extraction step to the amplification product detection step is performed. As a result, the present invention has been completed.

That is, the present invention
(1) To suppress variation in expression profile of a transformed cell introduced with an expression vector into which a standard nucleic acid having a known base sequence in whole or in part is introduced, while retaining the nucleic acid inside the cell. A method for producing a standard cell sample, characterized by having an inactivation treatment step of immersing in an inactivation liquid that can be produced,
(2) The mRNA according to (1), wherein mRNA synthesized using the standard nucleic acid as a template is present in the transformed cell, but no protein derived from the standard nucleic acid is produced. A standard cell sample production method of
(3) Before the inactivation treatment step,
Contacting an expression inducer to synthesize mRNA in the transformed cell using the standard nucleic acid as a template;
A method for producing a standard cell sample according to the above (1) or (2),
(4) The RNA transcribed from the region containing the standard nucleic acid in the expression vector is deficient in ribosome binding ability, as described in any one of (1) to (3) above A standard cell sample production method of
(5) The (1), wherein the inactivating liquid is a solution containing one or more selected from the group consisting of a water-soluble organic solvent, a protease inhibitor, a chelating agent, and salts as an active ingredient. A method for producing a standard cell sample according to any one of to (4),
(6) The method for producing a standard cell sample according to (5), wherein the water-soluble organic solvent is at least one selected from the group consisting of water-soluble alcohols, ketones, and aldehydes. ,
(7) The inactivating liquid contains a water-soluble alcohol and / or ketone as a water-soluble organic solvent, and the concentration of the water-soluble organic solvent is 30% or more (5) or (6) ) For producing a standard cell sample,
(8) The method for producing a standard cell sample according to (6) or (7), wherein the water-soluble alcohol is one or more selected from the group consisting of ethanol, propanol, and methanol,
(9) The method for producing a standard cell sample according to any one of (6) to (8), wherein the ketones are acetone and / or methyl ethyl ketone,
(10) The inactivation liquid contains an aldehyde as a water-soluble organic solvent, and the concentration of the water-soluble organic solvent is 0.01 to 30%, as described in (5) or (6) above A standard cell sample production method of
(11) The inactivating liquid contains sodium chloride at a concentration of 13% (wt / wt) or higher to a saturation concentration or lower thereof, according to any one of (5) to (10), A standard cell sample production method of
(12) The inactivating liquid contains ammonium sulfate at a concentration of 30% (wt / wt) or more to a saturation concentration or less thereof, according to any one of (5) to (11), Standard cell sample production method,
(13) The method for producing a standard cell sample according to any one of (5) to (12), wherein the inactivating liquid contains a chelating agent of 0.1 mM to 1M.
(14) After the inactivation treatment step,
A storage step of storing the transformed cells in a dispersion in an inactivated liquid at −20 to 40 ° C .;
The method for producing a standard cell sample according to any one of the above (1) to (13), comprising:
(15) The method for producing a standard cell sample according to any one of (1) to (14), wherein the standard nucleic acid is a nucleic acid derived from a human gene,
(16) The standard according to any one of (1) to (15), wherein the standard nucleic acid is a nucleic acid derived from a COX-2 (cyclooxygenase-2) gene or a nucleic acid derived from a c-Myc gene A method for producing a cell sample,
(17) The method for producing a standard cell sample according to any one of (1) to (16), wherein the transformed cell is a prokaryotic cell,
Is provided.

  According to the method for producing a standard cell sample of the present invention, it can be stably stored for a long period of time, and a series of extraction / purification of nucleic acid from a cell sample, amplification of the extracted nucleic acid, and detection of an amplification product In nucleic acid analysis having steps, a standard cell sample that can also be used for quality control of all steps can be easily produced.

It is the flowchart which showed the one aspect | mode of the analysis method of a nucleic acid. It is the flowchart which showed another one aspect | mode of the analysis method of a nucleic acid. In Example 2, it is the figure which showed the COX-2 gene expression level detected from the preserve | saved cell sample immersed in the ethanol solution of various density | concentrations. In Example 3, it is the figure which showed the COX-2 gene expression level detected from the standard cell sample preserve | saved at various temperature.

<< Standard cell sample and production method thereof >>
The standard cell sample produced by the method for producing the standard cell sample of the present invention (hereinafter sometimes referred to as the standard cell sample of the present invention) incorporates a standard nucleic acid having a known base sequence in whole or in part. An expression vector is introduced, and the transformed cell is immersed in an inactivating liquid that can suppress a change in expression profile while retaining a nucleic acid inside the cell. Since the cell expression profile is fixed by the action of the inactivating liquid, the standard cell sample of the present invention can be stably stored for a long period of time. In addition, after incorporating a standard nucleic acid into an expression vector and introducing the expression vector into cells by gene recombination techniques widely used in the art, the resulting transformed cells are converted into an inactivated liquid. It can be easily manufactured by a simple operation of dipping. Furthermore, the standard cell sample of the present invention uses a nucleic acid that can be used as a control for a nucleic acid amplification reaction as a standard nucleic acid to be incorporated into an expression vector, thereby extracting and purifying nucleic acid from the cell sample, and amplifying the extracted nucleic acid. And nucleic acid analysis having a series of steps of detection of amplification products, it can be suitably used as a standard sample for controlling the accuracy of all steps.

  In the present invention and the specification of the present application, “suppressing the fluctuation of the expression profile” means to stop and stabilize the increase or decrease of the gene expression level in the cell. The gene expression level can be measured by appropriately using a technique known in the technical field such as a real-time PCR method. For example, when the measurement value obtained by measuring the expression level of a specific gene in a cell sample is almost equal to the measurement value obtained in the same way at different time points, fluctuations in the expression profile of the cell sample are suppressed. It can be said that.

  The standard nucleic acid to be incorporated into the expression vector may be any nucleic acid as long as the whole or part of the base sequence is known to the extent that an amplification product can be obtained by a nucleic acid amplification reaction such as PCR. . In the present invention and the present specification, the nucleic acid includes both DNA and RNA, but the standard nucleic acid is DNA. In the present invention, the standard nucleic acid is preferably a nucleic acid containing a protein-coding region, and more preferably a nucleic acid containing a gene-derived nucleic acid. In addition, the nucleic acid derived from a gene is a genomic DNA or genomic RNA of an organism gene, RNA transcribed from the full length or part of the genomic DNA or genomic RNA, and a base sequence complementary to the full length or part of these nucleic acids. And the like. For example, not only the full length or a part of the genomic DNA of one gene, but also cDNA synthesized by reverse transcription reaction from the full length or a part of mRNA, amplification products obtained by amplifying the cDNA by PCR, etc. It is contained in the nucleic acid derived from.

  The gene-derived nucleic acid is preferably a human gene-derived nucleic acid, and particularly preferably a nucleic acid derived from a disease marker gene. A marker gene for a disease is to determine whether or not the subject suffers from the disease by analyzing the presence or absence of expression of the gene in the biological sample to be analyzed and the amount of the expression. Means a possible gene. Specifically, a disease marker gene includes a gene that is specifically expressed when suffering from a specific disease, a base insertion, deletion, substitution, duplication, inversion, or splicing variant (isotopic). Gene) in which a mutation occurs.

  Examples of disease marker genes include marker genes for adenomas and cancer, marker genes for infectious diseases, and the like. In diseases such as adenoma and cancer, gene mutation is considered to be one of the main causes of onset, and marker genes are detected by genetic analysis in clinical tests. Examples of adenoma or cancer marker genes include COX-2 (Cyclooxygenase-2), c-Myc gene, MMP7 (matrix metallopeptidase 7), SNAIL, and the like. Examples of infectious disease marker genes include genes of causative microorganisms for infectious diseases.

  In addition, the standard nucleic acid incorporated into the expression vector may be a nucleic acid containing a nucleic acid derived from a housekeeping gene. Housekeeping genes include, for example, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA, 28S ribosomal RNA, β actin, β2 microglobulin, hypoxanthine phosphoribosyl transferase 1, and ribosome Protein large P0, peptidylpropyl isomerase A (cyclosporin A), cytochrome C, phosphoglycerate kinase 1, β-glucuronidase, TATA box binding factor, transferrin receptor, HLA-A0201 heavy chain, ribosomal protein L19, α tubulin , Β-tubulin, γ-tubulin, ATP synthetase, translation elongation factor 1 gamma (EEF1) : Eukaryotic elongation factor 1 gamma), succinate dehydrogenase complex (SDHA), succinate dehydrogenase complex (DPAS), aminolevulinate synthase 1 (ALAS: aminolevulinic acidA) Examples thereof include nuclease G (ENDOG: endonclease G), and peroxisome formation factor (PEX).

  In the present invention, a cell into which an expression vector incorporating a standard nucleic acid is introduced may be a prokaryotic cell (bacteria) such as Escherichia coli or Bacillus subtilis, such as yeast, filamentous fungus, insect cell, or mammalian cell. It may be a eukaryotic cell. In the present invention, a bacterium such as Escherichia coli is preferable because it is easy to produce and has excellent handleability. Bacteria can be easily cultured in large quantities, and are stable to cryopreservation and freeze-thawing treatment. Furthermore, the introduced expression vector can be retained relatively stably. For this reason, it is possible to prepare and stock a large number of standard cell samples of the same lot by dispensing a suitable amount of bacterial culture solution prepared in large quantities at a time and storing them frozen. In addition, when mammalian cells such as humans are used, a standard cell sample having a human-derived gene expression profile close to a clinical specimen and a uniform and immobilized expression profile can be produced.

  As expression vectors, plasmid vectors, virus vectors, cosmid vectors, BAC vectors, λ phage vectors and the like are known. An expression vector into which a standard nucleic acid is incorporated can be appropriately selected from vectors known in the art depending on the cell type to be introduced. Further, a known vector may be modified by a gene recombination technique or the like. In the present invention, it is preferable to use a plasmid vector because it is easy to produce and has excellent handleability.

  The standard nucleic acid can be incorporated into the expression vector by a conventional method using a known gene recombination technique. In addition, the method of introducing the expression vector into cells can be appropriately selected from methods known in the art in consideration of the type of expression vector, the type of cell, and the like. Specifically, examples of the method for introducing a plasmid vector into a cell include an electroporation method, a calcium phosphate method, a liposome method, and a DEAE dextran method. Commercially available vector introduction reagents may also be used. In addition, retrovirus vectors, adenovirus vectors, adenovirus-associated vectors, and the like are widely used as virus vectors. In these cases, a virus vector into which a standard nucleic acid has been incorporated was introduced into a packaging cell. Viral vectors can be introduced into cells by contacting the cells with a recombinant virus for infection.

  When a vector that causes transcription / translation of a gene incorporated in the expression vector by inducing expression with a specific expression inducer is used as an expression vector, an expression vector incorporating a standard nucleic acid is used. The introduced transformed cell is preferably brought into contact with an expression inducer to induce expression before being immersed in an inactivating liquid. By fixing the expression profile in the state where mRNA is synthesized from the standard nucleic acid by induction of expression, the obtained standard cell sample is analyzed by analyzing the mRNA in the cell, such as gene expression level analysis. Therefore, it can be suitably used as a sample for process accuracy control.

  Expression induction can be performed by adding an expression inducer to the culture medium of transformed cells. Control the amount of RNA (standard nucleic acid-derived RNA) transcribed from the region containing the standard nucleic acid in the expression vector within the desired range by appropriately adjusting the type and amount of the expression inducer and the expression induction time. Alternatively, the amount of standard nucleic acid-derived RNA between transformed cells can be made uniform. That is, it becomes possible to regulate the expression profile of the gene in the transformed cell. Thus, when a standard cell sample is used as a sample for process accuracy control of nucleic acid analysis, the amount of standard nucleic acid-derived RNA contained in the standard cell sample is adjusted according to the type of target nucleic acid to be analyzed, etc. You can also

  In the present invention, it is preferable that a protein transcribed and translated from a standard nucleic acid introduced by an expression vector is not produced in a transformed cell. The expression profile may be affected by the presence of the protein derived from the standard nucleic acid in the cell, such as when the protein derived from the standard nucleic acid is toxic to the transformed cells. Since the degree of toxicity often varies from cell to cell, the expression profile may vary between transformed cells. In addition, when the protein derived from the standard nucleic acid forms an inclusion body in the cell, the extraction solution used for extracting the nucleic acid from the cell due to the presence of the protein derived from the standard nucleic acid in the cell. There may be an increase in the amount of insoluble components. Nucleic acid extraction efficiency may be affected by the amount of insoluble components, but the amount of insoluble components increased by foreign proteins often varies from cell to cell, so nucleic acid extraction efficiency varies between transformed cells. There is a fear. When a protein derived from a standard nucleic acid is not produced in the transformed cells, such a fear can be avoided, and the expression profile between the transformed cells and the nucleic acid extraction efficiency can be made more uniform.

  Even when the standard nucleic acid to be introduced into the expression vector is a nucleic acid encoding a protein such as a gene-derived nucleic acid, for example, the ribosome ability is preferably such that the ribosome-binding ability of the standard nucleic acid-derived RNA is significantly reduced. By modifying the expression vector in advance so that it is deleted, the expression of the protein derived from the standard nucleic acid can be suppressed. Specifically, a mutation that causes a ribosome binding site (RBS) upstream of the standard nucleic acid insertion site in the expression vector to be deleted or a ribosome binding ability to be reduced or deleted is introduced into the RBS. Examples of RBS of prokaryotic cells include SD (Shine Dalgarno) sequence and the like. In eukaryotic cells, the standard can also be obtained by deleting the Kozak sequence upstream of the standard nucleic acid insertion site in the expression vector, or by introducing a mutation that reduces or eliminates the translation promoting ability of the Kozak sequence. Expression of nucleic acid-derived protein can be suppressed. In addition, it is also preferable to introduce a standard nucleic acid excluding the start codon into the expression vector.

  A standard cell sample can be produced by immersing a transformed cell into which an expression vector incorporating a standard nucleic acid is introduced into an inactivation liquid. By soaking in an inactivating liquid, the synthesis and degradation reactions of nucleic acids and proteins in the transformed cells are stopped, and the expression profile is fixed. Inactivated liquid kills transformed cells and inactivates them, but keeps the nucleic acid retained in the cells, so it is derived from standard nucleic acids in expression vectors and standard nucleic acids synthesized by transcription RNA is stably retained in the cell.

  The inactivating liquid used in the present invention is a solution having an action effect (hereinafter referred to as an inactivating effect) that suppresses fluctuations in the expression profile while retaining the nucleic acid inside the cell. The inactivating liquid is preferably a solution containing one or more selected from the group consisting of water-soluble organic solvents, protease inhibitors, chelating agents, and salts as active ingredients. For example, a water-soluble organic solvent or a diluted solution thereof, or an aqueous solution having a high salt concentration may be used as the inactivating liquid. Alternatively, a protease inhibitor or a chelating agent alone or in combination and dissolved in a water-soluble organic solvent or a diluted solution thereof or an aqueous solution having a high salt concentration may be used as the inactivating liquid.

  In the present invention and the specification of the present application, the water-soluble organic solvent means an organic solvent having high solubility in water or miscible with water at an arbitrary ratio. Since cells have a high water content, they can be used as an active ingredient for inactivated liquid by using a solvent with high solubility in water or a water-soluble organic solvent that can be mixed with water at an arbitrary ratio. The activation liquid can be quickly mixed.

  Although the reason why the water-soluble organic solvent has an inactivating effect is not clear, the dehydration action of the water-soluble organic solvent component significantly reduces the cell activity of the transformed cells, and the water-soluble organic solvent It is presumed that the activity of various degrading enzymes such as protease, DNase, and RNase in the transformed cells is remarkably reduced due to the protein denaturing action of the component.

  Specific examples of the water-soluble organic solvent used as the inactivating liquid include alcohols, ketones, or aldehydes, which have a linear structure and are liquid at around room temperature, for example, 15 to 40 ° C. Means solvent. By using a water-soluble organic solvent having a linear structure as an active ingredient, mixing with cells can be performed more quickly than using an organic solvent having a cyclic structure such as a benzene ring as an active ingredient. Since organic solvents having a cyclic structure are generally easily separated from water, it is difficult to mix with cells having a high water content, and it is difficult to obtain a high inactivation effect. In order to facilitate mixing of the cells and the organic solvent having a cyclic structure, it may be possible to prepare a mixed solution of the organic solvent and water in advance and then mix the cells and the mixed solution. However, in order to prepare the mixed solution, it is often not necessary to vigorously mix or heat an organic solvent having a cyclic structure and water.

  The inactivating liquid used in the present invention is preferably a water-soluble organic solvent having a water solubility of 12% by weight or more, more preferably a water-soluble organic solvent having a water solubility of 20% by weight or more, A water-soluble organic solvent having a solubility in water of 90% by weight or more is more preferable, and a water-soluble organic solvent that can be mixed with water at an arbitrary ratio is particularly preferable. Examples of water-soluble organic solvents that can be mixed with water at an arbitrary ratio include methanol, ethanol, n-propanol, 2-propanol, acetone, formaldehyde, and the like.

  The water-soluble organic solvent used as the inactivating liquid in the present invention is not particularly limited as long as it satisfies the above definition and can exhibit an inactivating effect. Examples of the water-soluble organic solvent include alcohols such as methanol, ethanol, propanol, butanol, mercaptoethanol and the like as water-soluble alcohols, and ketones as acetone, methyl ethyl ketone (90% by weight solubility in water, etc.). And aldehydes include acetaldehyde (acetylaldehyde), formaldehyde (formalin), glutaraldehyde, paraformaldehyde, glyoxal, etc. The propanol may be n-propanol, 2-propanol The butanol may be 1-butanol (water solubility 20% by weight) or 2-butanol (water solubility 12.5% by weight), which is used in the present invention. Is The water-soluble organic solvent is preferably a water-soluble alcohol, acetone, methyl ethyl ketone, or formaldehyde because it has a sufficiently high solubility in water, from the viewpoints of availability, handleability, safety, etc. More preferred are ethanol, propanol, and methanol, and ethanol is particularly useful because it is the safest and can be handled easily.

  The concentration of the water-soluble organic solvent in the inactivating liquid is not particularly limited as long as it is a concentration that can exert an inactivating effect, and should be appropriately determined in consideration of the type of the water-soluble organic solvent. Can do. When the concentration of the water-soluble organic solvent in the inactivated liquid is sufficiently high, when the transformed cell and the inactivated liquid are mixed, the water-soluble organic solvent component rapidly penetrates the entire transformed cell, An effect can be produced quickly.

  For example, when using a water-soluble alcohol or ketone, the concentration of the water-soluble organic solvent in the inactivated liquid is preferably 30% or more, more preferably 50% or more, and 50 to 80%. Is more preferable, and 60 to 70% is particularly preferable. As the water-soluble organic solvent concentration is higher, a sufficient effect can be obtained by using a small amount of inactivating liquid for cells having a high water content.

  When acetone or methyl ethyl ketone is used, the concentration of the water-soluble organic solvent in the inactivated liquid is preferably 30% or more, more preferably 60% or more, and further preferably 80% or more. . In addition, when acetaldehyde, formaldehyde, glutaraldehyde, paraformaldehyde, or glyoxal is used as an active ingredient, the concentration of the water-soluble organic solvent in the inactivated liquid is preferably 0.01 to 30%. It is more preferably 03 to 10%, and further preferably 3 to 5%. Aldehydes can exert an inactivation effect even at a lower concentration than alcohols and ketones.

  In addition, the inactivating liquid used in the present invention may contain only one type of water-soluble organic solvent, or may be a mixed solution of two or more types of water-soluble organic solvents. For example, it may be a mixed solution of two or more kinds of alcohols, or a mixed solution of alcohols and other kinds of water-soluble organic solvents. Since the inactivation effect is further improved, a mixed solution of alcohol and acetone is also preferable.

  Moreover, in this invention, it is preferable to use a protease inhibitor as an active ingredient as an inactivation liquid. As a result of the degradation of cell membrane proteins and the like by proteases present inside and outside the cell, the nucleic acid flows out of the cell from pores or the like formed in the cell membrane, and the nucleic acid that flows out of the cell is nucleolytic enzyme that exists outside the cell. It will be decomposed by the action of. By using a protease inhibitor as the active ingredient of the inactivation liquid, the degradation of cell membrane proteins is effectively suppressed, and the expression profile of transformed cells is maintained by maintaining nucleic acids in cells with relatively small amounts of degrading enzymes. Fluctuations can be suppressed.

  In the present invention, the protease inhibitor used as the active ingredient of the inactivating liquid is not particularly limited as long as it can inhibit the enzyme activity of protease (protease, an enzyme that can catalyze the hydrolysis of peptide bonds). Instead, it may be a proteinase inhibitor or a peptidase inhibitor. Further, it may be one that can inhibit serine protease, one that can inhibit cysteine protease, or one that can inhibit aspartic protease (acidic protease), It may be capable of inhibiting a metalloprotease.

  The protease inhibitor used in the present invention can be appropriately selected from known protease inhibitors. Examples of protease inhibitors used in the present invention include AEBSF, Aprotinin, Bestin, Calpain Inhibitor 1, Calpain Inhibitor II, Chymostatin, 3,4-Dichloroisocomain, E-64, Lactacytin, G, , Pepstatin A, PMSF, Proteasome Inhibitor, TLCK, TPCK, Trypsin Inhibitor, and the like. In addition, a combination of a plurality of types of protease inhibitors generally called “protease inhibitor cocktail” can also be used.

  Further, the concentration of the protease inhibitor in the inactivation liquid is not particularly limited as long as it is sufficient to inhibit the protease in the solution containing the transformed cells. Appropriately determined in consideration of the type, the mixing ratio of the solution containing the transformed cells and the inactivated liquid, the pH and temperature of the suspension prepared by mixing the solution containing the transformed cells and the inactivated liquid, etc. can do. Table 1 describes preferred concentrations of each protease inhibitor in a suspension prepared by mixing with a solution containing transformed cells.

  The protease inhibitor used in the present invention may be a peptide protease inhibitor as described above, a reducing agent, or a protein denaturing agent. In the present invention, the “peptide protease inhibitor” means a peptide capable of inhibiting protease activity by interacting with a protease, or a modified form thereof.

Examples of the reducing agent include DTT (dithiothreitol) and β-mercaptoethanol.
The concentration of the reducing agent added to the inactivating liquid as a protease inhibitor is not particularly limited as long as it is a sufficient concentration to inhibit the protease in the solution containing the transformed cells. It can be appropriately determined in consideration of the above. Preferably, each reducing agent is added so that the final concentration in a suspension prepared by mixing with a solution containing transformed cells is 0.1 mM to 1 M.

Examples of protein denaturing agents include urea, guanine, guanidine salts and the like.
The concentration of the protein denaturing agent added to the inactivating liquid as a protease inhibitor is not particularly limited as long as it is a concentration sufficient to inhibit the protease in the solution containing the transformed cells. It can be determined appropriately in consideration of the type of the above. Preferably, each protein denaturant is added so that the final concentration in a suspension prepared by mixing with a solution containing transformed cells is 0.1 mM to 10 M.

  In addition, as a nucleic acid stabilizer, only 1 type of protease inhibitor may be used and 2 or more types of protease inhibitors may be used. Also, a plurality of types of peptide protease inhibitors such as AEBSF may be used in combination, and different types of protease inhibitors may be used, such as peptide protease inhibitors and reducing agents.

  Moreover, in this invention, it is also preferable to use a chelating agent as an active ingredient of an inactivation liquid. The chelating agent can suppress enzyme activities such as nucleolytic enzymes and proteolytic enzymes inside and outside the transformed cells.

As a chelating agent, ethylenediaminetetraacetic acid (EDTA), O, O′-bis (2-aminophenylethyleneglycol) ethylenediaminetetraacetic acid (BAPTA), N, N-bis (2-hydroxyethyl) glycine (Bicine), Trans-1 , 2-Diaminocyclohexane-ethylenediaminetetraacetic acid (CyDTA), 1,3-diamino-2-hydroxybropan-ethylenediaminetetraacetic acid (DPTA-OH), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminedipropanoic acid hydrochloride (EDDP), ethylenediamine Dimethylenephosphonic acid monohydrate (EDDPO), N- (2-hydroxyethyl) ethylenediaminetriacetic acid (EDTA-OH), ethylenediaminetetramethylenephosphonic acid (EDTPO), O, O′-bis (2-amino) Til) ethylene glycol tetraacetic acid (EGTA), N, N-bis (2-hydroxybenzyl) ethylenediaminediacetic acid (HBED), 1,6-hexamethylenediaminetetraacetic acid (HDTA), N- (2-hydroxyethyl) imino Diacetic acid (HIDA), iminodiacetic acid (IDA), 1,2-diaminopropanetetraacetic acid (Methyl-EDTA), nitrilotriacetic acid (NTA), nitrilotripropanoic acid (NTP), nitrilotrimethylenephosphonic acid trisodium salt (NTPO), ethylenediaminetetra (2-pyridylmethyl) (TPEN), and triethylenetetraamine hexaacetic acid (TTHA).
If the concentration of the chelating agent added to the inactivating liquid as an active ingredient is sufficient to inhibit the enzyme activity in the solution containing the transformed cells and suppress the variation in the expression profile of the transformed cells, It is not particularly limited, and can be appropriately determined in consideration of the type of chelating agent. Preferably, each chelating agent is added so that the final concentration in a suspension prepared by mixing with a solution containing transformed cells is 0.1 mM to 1 M.

  Furthermore, a high salt concentration solution may be used as the inactivating liquid. The reason why a high salt concentration solution can function as an inactivating liquid is not clear, but various degrading enzymes are precipitated by salting out. Since the cell activity of bacteria such as bacteria is significantly reduced and changes over time are suppressed, and the activity of various degrading enzymes such as protease, DNase, and RNase is significantly reduced due to deviation from the optimum salt concentration. It is guessed.

  As a salt contained as an active ingredient in an inactivating liquid, it can select from the salt normally used when preparing or analyzing a biological sample, and can use it selecting it suitably. For example, it may be a hydrochloride, a sulfate, or an acetate. Moreover, as an active ingredient, one type of salt may be used, or two or more types of salts may be used in combination. The inactivating liquid used in the present invention includes sodium chloride, potassium chloride, ammonium sulfate, ammonium bisulfate, ammonium chloride, ammonium acetate, cesium sulfate, cadmium sulfate, cesium iron (II), chromium (III) sulfate as active ingredients. From the group consisting of cobalt sulfate (II), copper sulfate (II), lithium chloride, lithium acetate, lithium sulfate, magnesium sulfate, manganese sulfate, sodium sulfide, sodium acetate, sodium sulfate, zinc chloride, zinc acetate, and zinc sulfate It is preferable to include one or more selected. Especially, it is preferable that it is a solution containing sodium chloride and / or ammonium sulfate from points, such as availability, a handleability, and safety. Sodium chloride is particularly useful because it is the safest and can be handled easily.

  The concentration of the salt contained as an active ingredient in the inactivating liquid (hereinafter sometimes simply referred to as “salt concentration”) is not particularly limited as long as it is a concentration that functions as an inactivating liquid. It can be appropriately determined in consideration of the type of solvent. In each salt, the upper limit value of the salt concentration is the saturation concentration. On the other hand, although the lower limit value varies depending on the type of salt used, those skilled in the art can obtain it experimentally in advance.

For example, the lower limit value can be obtained as follows. First, salt solutions having a plurality of concentrations below the saturation concentration are prepared, and nucleic acids are collected from cells immersed in these salt solutions for a predetermined period.
The minimum salt concentration value when the amount of recovered nucleic acid is larger than the amount of nucleic acid recovered from cells that have not been subjected to salt solution treatment can be the lower limit value of the salt concentration of the salt contained as the active ingredient.

  In the present invention, in order to obtain a higher inactivation effect, regardless of the type of salt, the salt concentration of the inactivation liquid may be at least half the saturation concentration of the salt as the active ingredient. The concentration is preferably 4/5 or more of the saturated concentration, more preferably close to the saturated concentration, and particularly preferably substantially the saturated concentration. When the salt concentration is sufficiently high, when the cells are mixed with an inactivated liquid having a high salt concentration, the components can rapidly penetrate into the cells and the nucleic acid can be stabilized quickly. In addition, the higher the salt concentration, the more satisfactory effect can be obtained even when a small amount of inactivating liquid is used for cells having a high water content. In addition, “a solution having a concentration of ½ times the saturated concentration” and “a solution having a concentration of 4/5 or more of the saturated concentration” are obtained by appropriately diluting a saturated solution prepared by a conventional method with a solvent, for example. Can be prepared.

  For example, when sodium chloride is used as an active ingredient, the salt concentration in the inactivated liquid is preferably 13% (wt / wt) or more, more preferably 20% (wt / wt) or more, The concentration is more preferably 26% (wt / wt) or more, and particularly preferably from 26% (wt / wt) to the saturation concentration. When ammonium sulfate is used, the salt concentration is preferably 20% (wt / wt) or more, more preferably 30% (wt / wt) or more, and 30 to 46% (wt / wt). More preferably.

  The inactivating liquid used in the present invention can be adjusted to a desired concentration by diluting the active ingredient with an appropriate solvent. The solvent used for diluting the active ingredient is not particularly limited as long as it does not impair the effect of the active ingredient, that is, the effect of suppressing the variation in the expression profile of the transformed cell. For example, water or a buffer solution such as PBS may be used.

  The inactivating liquid used in the present invention is preferably a solution obtained by diluting an active ingredient, particularly a water-soluble organic solvent, with a buffer having a pH of 2 to 7.5, and the pH is within a range of 2 or more. More preferably, the solution is diluted with an acidic buffer having a buffering action that is maintained. The pH of the acidic buffer is preferably 2 to 6.5, more preferably 3 to 6, still more preferably 3.5 to 5.5, and 4.0 to 5.0. It is particularly preferred. By maintaining the transformed cell under acidic conditions, hydrolysis of the nucleic acid in the transformed cell can be more effectively suppressed.

  The acidic buffer used for diluting the active ingredient during the preparation of the inactivating liquid contains an organic acid and a conjugate base of the organic acid, and is buffered by the organic acid and the conjugate base. It is preferable that Among these, a buffer solution selected from the group consisting of a citric acid / sodium hydroxide buffer system, a lactic acid / sodium lactate buffer system, and an acetic acid / sodium acetate buffer system is preferable. Such a buffer solution can be prepared by adjusting to a desired pH by adding an organic acid and an alkali metal salt or alkaline earth metal salt of the organic acid to water or an appropriate solvent, for example. In addition, after adding an organic acid to water or a suitable solvent, the pH may be adjusted using an alkali metal or alkaline earth metal hydroxide.

  In addition, the acidic buffer used for diluting the active ingredient to prepare the inactivated liquid is a solution containing both an organic acid and a mineral acid, and may have an appropriate buffering action. . For example, a buffer system having a buffering action on the acidic side, such as a glycine / HCl buffer system, a cacodylate Na / HCl buffer system, or a phthalate HK / HCl buffer system may be used.

  In the present invention and the specification of the present application, the pH of the acidic buffer solution is determined by using a pH meter (for example, manufactured by Toa DKK Corporation) based on the glass electrode method as a measurement principle using a phthalate standard solution and a neutral phosphate standard solution. It is a value obtained by measuring after calibration.

  In addition, for example, chaotropic salts, surfactants, polycations and the like may be added to the inactivating liquid used in the present invention as long as the inactivating effect is not impaired.

  By adding a chaotropic salt or a surfactant to the inactivating liquid, it is possible to more effectively inhibit the cell activity of transformed cells and the enzyme activities of various degrading enzymes. Examples of chaotropic salts that can be added to the inactivating liquid include guanidine hydrochloride, guanidine isothiocyanate, sodium iodide, sodium perchlorate, and sodium trichloroacetate. The surfactant to be added to the inactivating liquid is preferably a nonionic surfactant. Examples of the nonionic surfactant include Tween 80, CHAPS (3- [3-colamidopropyldimethylammonio] -1-propanesulfonate), Triton X-100, Tween 20, and the like. The concentration of the chaotropic salt or the surfactant is not particularly limited as long as the inactivation effect is obtained. Considering the amount of transformed cells and the method of using the produced standard cell sample, etc. Can be determined as appropriate.

  In the present invention and the present specification, polycation means a polymer compound having a repeating structure containing a cationic functional group and a salt thereof. Examples of the cation include an amino group. Specifically, it is obtained by polymerizing a polypeptide having a cationic functional group in the side chain such as polylysine represented by the following formula (1) or a monomer having a cationic functional group such as polyacrylamide in the side chain. And the like. These polypeptides and polymers need only be electrically positive as a whole molecule and do not need to have a cationic functional group in the side chain of all repeating units (amino acids and monomers). It is preferable to have a cationic functional group in the side chain of all repeating units. Specifically, in addition to polylysine and polyacrylamide, such polycations include polyvinylamine, polyallylamine, polyethylamine, polymethallylamine, polyvinylmethylimidazole, polyvinylpyridine, polyarginine, chitosan, 1,5-dimethyl. -1,5-diazaundecamethylene-polymethobromide, poly (2-dimethylaminoethyl (meth) acrylate), poly (2-diethylaminoethyl (meth) acrylate), poly (2-trimethylammoniumethyl (meth)) Acrylate), polydimethylaminomethylstyrene, polytrimethylammonium methylstyrene, polyornithine, polyhistidine and the like. In the present invention, the polycation is preferably polylysine or polyacrylamide, and more preferably polylysine. In addition, when adding to an inactivation liquid, only 1 type of polycation may be added and 2 or more types of polycation may be added.

The concentration of the polycation added to the inactivation liquid is not particularly limited as long as the inactivation effect is obtained, and the type of polycation, the type of transformed cell, the pH of the inactivation liquid, It can be appropriately determined in consideration of the mixing ratio between the inactivation effect and the solution containing the transformed cells. For example, when polylysine is added as a polycation, the polylysine concentration of the inactivating liquid is preferably 0.01 to 1.0 m% by weight, more preferably 0.0125 to 0.8 m% by weight. Preferably, it is 0.05 to 0.4 m% by weight. In the present specification, “m wt%” means “× 10 ⁻³ wt%”.

  The time for immersing the transformed cell in the inactivation liquid is not particularly limited as long as it is sufficient for the inactivation effect to be achieved. Depending on the composition of the inactivation liquid, the type of the transformed cell, etc. Can be determined as appropriate. In the present invention, it is preferably immersed for 1 hour or longer, more preferably 12 hours or longer, further preferably 24 hours or longer, particularly preferably 72 hours or longer. Moreover, you may immerse for 168 hours or more.

  Since the expression profile of the standard cell sample of the present invention is fixed by the inactivating liquid, it is stable for a long time at −20 to 40 ° C. in the state of the dispersion dispersed in the inactivating liquid without coagulation. Can be saved. Easy to handle because it can be stored at room temperature. In addition, since the standard cell sample can be stored in the state of a dispersion, it is not necessary to prepare a sample such as a lysis treatment before being subjected to nucleic acid analysis, and the working time can be shortened. The stored standard cell sample may be subjected to nucleic acid analysis in the same manner as the cell sample to be analyzed while immersed in an inactivated liquid, separated from the inactivated liquid and dispersed in an appropriate buffer. Then, it may be used for nucleic acid analysis.

<< Nucleic acid analysis method >>
The standard cell sample produced as described above is suitably used for quality control of nucleic acid analysis having a series of steps of extraction / purification of nucleic acid from the cell sample, amplification of the extracted nucleic acid, and detection of the amplification product. be able to. The standard cell sample of the present invention can be subjected to a series of steps in the same manner as the cell sample to be analyzed, and can be used as a standard sample for all steps. Specifically, nucleic acid extraction efficiency from the standard cell sample of the present invention, the state of the extracted nucleic acid, the presence or absence of nucleic acid amplification from the extracted nucleic acid, the amplification efficiency, the amount of amplification product, etc. are correctly performed. Can be used as an accuracy indicator.

  In the present invention and the present specification, the target nucleic acid is a nucleic acid to be analyzed, and all or part of the base sequence is known to the extent that an amplification product can be obtained by a nucleic acid amplification reaction such as PCR. Any nucleic acid may be used. In the present invention, the target nucleic acid is preferably a nucleic acid containing a protein-encoding region, and more preferably a nucleic acid containing a gene-derived nucleic acid. The gene-derived nucleic acid is preferably a nucleic acid derived from a human gene, and more preferably a nucleic acid derived from a disease marker gene. Examples of disease marker genes include the same genes listed as standard nucleic acids. In addition, it is also preferable to use a gene-derived nucleic acid containing a gene polymorphism such as SNP as a target nucleic acid.

  The target nucleic acid and the standard nucleic acid may be completely different nucleic acids, or may be nucleic acids having the same full-length or partial base sequence. Further, the target nucleic acid and the standard nucleic acid may be nucleic acids derived from the same biological species, or may be nucleic acids of different biological species. As a standard cell sample used in the nucleic acid analysis method, the standard nucleic acid is preferably a nucleic acid derived from the same biological species as the target nucleic acid, and more preferably has the same base sequence as the target nucleic acid. The reaction conditions and reaction efficiency of nucleic acid amplification reactions such as PCR often differ depending on the type of primer used. For this reason, when the target nucleic acid and the standard nucleic acid have the same base sequence, nucleic acid amplification is performed on the nucleic acid extracted from the cell sample to be analyzed by using a primer designed for the region where this base sequence is common. The reaction conditions and reaction efficiency of the nucleic acid amplification reaction for the reaction and the nucleic acid extracted from the standard cell sample can be made uniform, which is more preferable in terms of process accuracy control.

  Conventionally, when performing process accuracy control from nucleic acid extraction to nucleic acid amplification reaction and subsequent amplification product detection process using a single standard sample, an appropriate clinical sample for each target nucleic acid type is used as the standard sample. It was necessary to use it. For example, when the target nucleic acid is a nucleic acid expressed in a tissue-specific manner, it is necessary to use a clinical sample derived from the tissue as a standard sample. In contrast, in the present invention, the type of standard nucleic acid may be an appropriate nucleic acid for each target nucleic acid, and the cells into which the standard nucleic acid is introduced can be made common regardless of the type of target nucleic acid. For this reason, when analyzing a plurality of target nucleic acids, a standard sample can be more easily prepared.

  The cell sample to be analyzed to be subjected to the nucleic acid analysis method may be a sample (biological sample) collected from a living organism, or a sample prepared from cultured cells or the like. The cell sample used in the present invention is preferably a biological sample such as a clinical specimen. The organism from which the biological sample is collected is not particularly limited, but is preferably a eukaryote, more preferably an animal, still more preferably a mammal, and particularly preferably a human. . Examples of the biological sample include feces, urine, blood, bone marrow fluid, lymph fluid, sputum, saliva, semen, bile, pancreatic fluid, ascites, exudate, amniotic fluid, intestinal lavage fluid, lung lavage fluid, bronchial lavage fluid, bladder lavage fluid, oral mucosa Or uterine mucosa.

Specifically, the target nucleic acid analysis method includes the following steps (a) to (d).
(A) a step of extracting nucleic acid from a cell sample to be analyzed;
(B) amplifying a target nucleic acid by a nucleic acid amplification reaction using the nucleic acid extracted in the step (a) or a nucleic acid synthesized using the nucleic acid as a template as a template, and detecting the obtained amplification product;
(C) An expression vector into which a standard nucleic acid having a known base sequence in whole or in part has been introduced, and fluctuations in the expression profile can be suppressed while retaining the nucleic acid inside the cell. A step of extracting nucleic acid from a standard cell sample composed of transformed cells immersed in an activation liquid;
(D) A step of amplifying a standard nucleic acid by a nucleic acid amplification reaction using the nucleic acid extracted in the step (c) or a nucleic acid synthesized using the nucleic acid as a template as a template, and detecting the obtained amplification product.
Based on the result of the step (c) and / or (d), the process accuracy of the step (a) and / or (b) can be managed, and the reliability of the obtained result can be evaluated.

  The method for extracting nucleic acid from the cell sample or standard cell sample in steps (a) and (c) is not particularly limited, and any method known in the art may be used. For example, as a method for extracting and purifying RNA from cells, an acidic phenol guanidine-chloroform method such as ISOGEN, a boom method and the like can be mentioned, and these methods can be used in combination. In addition, commercially available purification kits can also be used.

  In addition, after RNA is extracted from a cell sample to prepare an RNA solution, the RNA solution is normalized (adjusted to a predetermined concentration) before proceeding to steps (b) and (d). Also good. The concentration to be normalized can be appropriately determined in consideration of the subsequent nucleic acid amplification reaction, detection method of the amplification product, and the like.

  When the nucleic acid extracted in steps (a) and (c) is DNA, in steps (b) and (d), a nucleic acid amplification reaction is performed using the extracted DNA as a template. When the nucleic acid extracted in steps (a) and (c) is RNA, in steps (b) and (d), a nucleic acid amplification reaction may be performed using the extracted RNA as a direct template. Alternatively, a reverse transcription reaction may be performed using the prepared RNA as a template, and a nucleic acid amplification reaction may be performed using the synthesized cDNA as a template.

  In the steps (b) and (d), a method of performing a reverse transcription reaction on the total amount or a part of the RNA in the RNA solution obtained by the steps (a) and (c) is also commonly used reverse transcriptase, etc. The reaction can be carried out under the reaction conditions generally used.

  The nucleic acid amplification method performed in steps (b) and (d) is not particularly limited as long as it is a method for amplifying a nucleic acid having a specific base sequence using DNA or RNA as a template. Any method known in the art may be used. For example, PCR, LAMP (Loop-Mediated Isothermal Amplification), may be a ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids), NASBA (Nucleic Acid Sequence-Based Amplification) and TRC (Transcription Reverse-transcription Concerted reaction Or the like.

  In the steps (b) and (d), the method for detecting the obtained amplification product is not particularly limited as long as it is a method that can generally detect DNA or RNA, and is a qualitative detection method. It may be a quantitative detection method. Examples of such detection methods include electrophoresis, hybridization, immunoaggregation, ELISA, absorbance measurement, single molecule fluorescence analysis, and fluorescence polarization analysis. Since it is possible to easily detect and quantify a small amount of nucleic acid in a sample, it is preferable to measure using a quantitative nucleic acid amplification method, ELISA method, single molecule fluorescence analysis method, or fluorescence polarization analysis method . For example, by performing real-time PCR, real-time NASBA, or the like, nucleic acid amplification reaction and amplification product detection can be performed simultaneously.

  In the target nucleic acid analysis method, the result obtained using the standard cell sample is used for accuracy control of each operation. Therefore, step (a) and step (c), step (b) and step (d) are performed under substantially the same conditions. That is, the nucleic acid extraction method in step (c) performs the same operation as in step (a) except that a standard cell sample is used instead of the cell sample to be analyzed. The nucleic acid amplification reaction and the amplification product detection method in step (d) are the same as in step (b) except that the nucleic acid extracted in step (c) is used instead of the nucleic acid extracted in step (a). Perform almost the same operation. Here, substantially the same conditions mean substantially the same conditions. For example, when the base sequences of the standard nucleic acid and the target nucleic acid are different, the primers used for nucleic acid amplification are designed so that the conditions such as the temperature of each nucleic acid amplification reaction are substantially the same. In the present invention, it is particularly preferable that the primers used in the nucleic acid amplification reaction in step (b) and the nucleic acid amplification reaction in step (d) are the same.

  Steps (a) and (c) may be performed at different times, but are preferably performed at substantially the same time. Similarly, steps (b) and (d) may be performed at different times, but are preferably performed at substantially the same time. Note that “substantially the same time” means substantially the same time.

  In addition, the steps (a) and (b) can also be performed by mixing a cell sample and a standard cell sample and then extracting a nucleic acid from the obtained mixture. In this case, in the subsequent nucleic acid amplification reactions of steps (b) and (d), the standard nucleic acid and the target nucleic acid may be used with different primer sets, but the same primer set can also be used. For example, one primer set is used from a nucleic acid extracted from a mixed solution of a cell sample and a standard cell sample by replacing a few bases in the target nucleic acid and creating a restriction enzyme site as a standard nucleic acid. Even when PCR is performed, by treating the obtained product with a restriction enzyme, the amplification product derived from the target nucleic acid and the amplification product derived from the standard nucleic acid can be distinguished and detected.

  In the steps (a) and (c), when the nucleic acid extraction operation is performed accurately, nucleic acid with sufficient quantity and quality is extracted from the standard cell sample. Therefore, the amount and quality of the nucleic acid extracted from the standard cell sample is examined, and when these satisfy a predetermined standard, it can be determined that the accuracy of the extraction step (step (a)) is guaranteed. it can. Conversely, when the quantity or quality of the nucleic acid extracted from the standard cell sample does not meet the preset standard, the accuracy of the step (a) is not guaranteed, and the nucleic acid extracted by the step (a) is not guaranteed. It can be determined that the result of the step (b) performed using the nucleic acid is low in reliability.

  In the specification of the present application, “the accuracy of the process is guaranteed” means that the operation of the process is appropriate, and there is no problem in operation. Conversely, “the accuracy of the process is not guaranteed” means that a malfunction has occurred due to deterioration of the reagent used, failure of the instrument or device, human error, etc., and the process is properly operated. It means that it was not.

  Specifically, one or more selected from the group consisting of the amount of nucleic acid extracted from the standard cell sample in step (c), the degree of purification, and the degree of degradation are measured. The accuracy of the process may be guaranteed based on the measured value of only one of the amount of nucleic acid, the degree of purification, or the degree of degradation, or the accuracy of the process may be guaranteed by combining the results of two or more measured values. The accuracy of the process may be guaranteed based on the results of all three types of measurement values.

  The amount of the extracted nucleic acid can be calculated from the nucleic acid concentration in the nucleic acid solution. The nucleic acid concentration in the nucleic acid solution can be quantified by a commonly used method. For example, the concentration can be calculated based on an ultraviolet absorption value at 260 nm, a fluorescence value measured using a double-stranded nucleic acid binding substance such as an intercalator, and the like. When the amount of the extracted nucleic acid is smaller than a preset threshold value, it is determined that the accuracy of the step (a) is not guaranteed. When the extracted nucleic acid amount is larger than the preset threshold value, the step (a ) Is guaranteed to be guaranteed. In this case, the threshold value serving as a judgment criterion can be appropriately determined in consideration of the amount of standard cell sample used for extraction, the amount of standard nucleic acid in the standard cell sample, the nucleic acid quantification method, and the like. For example, the threshold value is a measured value of the amount of nucleic acid extracted in each trial when nucleic acids are extracted multiple times from a standard cell sample of a fixed amount of the same lot (prepared simultaneously and subdivided). From the values obtained by statistical processing. The threshold value may be determined empirically.

The purity of nucleic acid means the ratio of impurities (substances other than nucleic acids) in the extracted and purified nucleic acid A. The higher the purity of the nucleic acid, the higher the quality of the nucleic acid.
The measurement of the purity of the nucleic acid can be performed by appropriately selecting from known methods generally used for measuring the purity (purity) of a nucleic acid sample. Among them, the absorbance of RNA was measured using UV, and the value obtained by dividing the absorbance at 260 nm by the absorbance at 230 nm (260/230 nm absorbance ratio) and the value obtained by dividing the absorbance at 260 nm by the absorbance at 280 nm (260/280 nm absorbance ratio). Is preferably used as an index of the degree of purification. From the 260/230 nm absorbance ratio, the concentration ratio between nucleic acid and salt can be determined. On the other hand, since the concentration ratio between nucleic acid and protein is known from the 260/280 nm absorbance ratio, the purity of the nucleic acid can be known from these. That is, when the 260/230 nm absorbance ratio or the 260/280 nm absorbance ratio is out of the preset numerical range, it can be determined that the accuracy of the step (a) is not guaranteed. Either of these absorbance ratios may be used, or both may be used.

  Specifically, when the 260/230 nm absorbance ratio is less than 1.0 or more than 2.5, the salt content is high, the degree of purification is insufficient, and the accuracy of the extraction process is not guaranteed. It can be judged. Conversely, when the 260/230 nm absorbance ratio is 1.0 to 2.5, preferably 1.7 to 2.1, the degree of purification is sufficient and the accuracy of step (a) is guaranteed. Can be determined. On the other hand, if the 260/280 nm absorbance ratio is less than 1.0 or more than 2.5, there is protein contamination and the like, and the degree of purification is considered to be insufficient, and the accuracy of step (a) is guaranteed. It can be judged that it is not. Conversely, when the 260/280 nm absorbance ratio is 1.0 to 2.5, preferably 1.7 to 2.1, the degree of purification is sufficient, and the accuracy of step (a) is guaranteed. Can be determined.

In the present invention, the degree of nucleic acid degradation means the rate at which the extracted and purified nucleic acid is degraded by a nucleolytic enzyme or the like. The lower the degree of nucleic acid degradation, the higher the quality of the nucleic acid.
Measurement of the degree of nucleic acid degradation can be performed by appropriately selecting from known methods generally used for measuring the degradation and fragmentation of nucleic acids. For example, when size separation measurement is performed by electrophoresis of nucleic acids, the amount of nucleic acid for each size can be known, so the degree of nucleic acid degradation can be measured.

  When a transformed bacterium is used as a standard cell sample and RNA is extracted in steps (a) and (c), the bacterium-derived RNA, particularly 23S rRNA-16S rRNA subunit, which is a ribosomal RNA of the bacterium, is used as an indicator. It is effective to measure the degradation degree of RNA. For example, in total RNA that has not been degraded, distinct bands of two ribosomal RNAs (bacteria-derived 23S rRNA and 16S rRNA) are seen at a ratio of approximately 2: 1. On the other hand, in total RNA in which degradation / fragmentation has occurred, the bands of each subunit of ribosomal RNA diffuse, the bands become unclear, and are detected in a smear state at a low molecular size. For this reason, when the 23S rRNA / 16S rRNA ratio is out of the preset numerical range, it can be determined that the accuracy of the step (a) is not guaranteed.

  Specifically, when the value obtained by dividing the fragment amount of 23S ribosomal RNA by the fragment amount of 16S ribosomal RNA (23S rRNA / 16S rRNA ratio) is 1.6 to 2.5, preferably 1.8 to 2. In the case of 0, it can be determined that the degree of decomposition is sufficiently low and the accuracy of the step (a) is guaranteed. Conversely, when the 23S rRNA / 16S rRNA ratio is less than 1.6 or more than 2.5, it can be determined that the degree of degradation is high and the accuracy of the step (a) is not guaranteed.

  The electrophoresis device “Bioanalyzer” manufactured by Agilent Technologies, which can be used for electrophoresis of RNA, is one of automatic capillary gel electrophoresis devices widely used in the field of molecular biology (for example, “A microfluidic system”). for highspeed reproducible DNA sizing and quantitation ", Electrophoresis, 200, Vol. 21, No. 1, pp. 128-134). This is because the quantification result for each size of nucleic acid is automatically displayed after the measurement is completed, so that the ribosomal RNA ratio is the 28S rRNA / 18S rRNA ratio (the value obtained by dividing the 28S ribosomal RNA fragment amount by the 18S ribosomal RNA fragment amount). 23S rRNA / 16S rRNA ratio and other band values are known, and the degradation / purification degree of the target RNA can be estimated from the ratio of degradation / fragmentation of ribosomal RNA. An RIN (RNA Integrity Number) value, which is one of the algorithms of this apparatus, is generally used as one of indicators of the degree of degradation of nucleic acids. When this RIN value (range: 1 to 10) is used, it can be said that the degree of decomposition is small when the RIN value is high (= 10), and the degree of decomposition is high when the RIN value is low (= 1). For example, in the RNA derived from a transformed bacterium, if the RIN value ranges from 10 to 4, it can be determined that the degree of degradation is sufficiently low.

  In the steps (b) and (d), when the nucleic acid amplification reaction is appropriately performed, the nucleic acid amplification product of the expected amount and quality is detected from the nucleic acid extracted from the standard cell sample. Therefore, the accuracy control of the nucleic acid amplification / detection step (step (b)) can be performed using the amount, purity, and amplification efficiency of the amplification product obtained from the nucleic acid extracted from the standard cell sample as indicators. Specifically, the amount, purity, and amplification efficiency of the amplification product obtained in step (d) are examined, and if these satisfy preset criteria, the accuracy of step (b) is guaranteed. It can be judged. On the contrary, when the amount of the amplification product obtained from the nucleic acid extracted from the standard cell sample does not satisfy a preset standard, the accuracy of the step (b) is not guaranteed, and the step (b It can be judged that the reliability of the result obtained by (1) is low.

  When RNA is extracted in steps (a) and (c), and a nucleic acid amplification reaction is performed in steps (b) and (d) using cDNA obtained after reverse transcription as a template, in step (d) The accuracy control of the step (b) can be performed using one or more selected from the group consisting of the amount of the detected amplification product, the purity of the amplification product, the amplification efficiency, and the reverse transcription reaction efficiency as an index.

  The amount, purity, amplification efficiency, and reverse transcription reaction efficiency of the amplification product are determined by using statistical data such as the standard deviation calculated from the detection measurement value after the reverse transcription reaction and the detection measurement value of the amplification product. be able to.

  The amount and purity of the amplification product can be appropriately selected from known methods usually used for measuring the amount and purity of the nucleic acid. Specifically, it can be performed in the same manner as the measurement of the amount and purity of the extracted nucleic acid. The amplification efficiency can be measured or calculated by detecting the amplification product quantitatively or semi-quantitatively. The amount of amplification product and the amplification efficiency can also be measured by performing a nucleic acid amplification reaction using a semi-quantitative amplification method such as real-time PCR. Furthermore, the reverse transcription reaction efficiency can be calculated from the amount, purity and the like of the reverse transcription reaction product.

  The threshold value used as a criterion is appropriately determined in consideration of the amount of nucleic acid used as a template for the reverse transcription reaction or nucleic acid amplification reaction, the type and reaction conditions of the primer used, the detection method of the reverse transcription reaction product or amplification product, etc. be able to. For example, the threshold value is a measured value of the product detected in each trial by performing reverse transcription reaction and nucleic acid amplification reaction multiple times under the same conditions using a certain amount of nucleic acid extracted from a standard cell sample as a template. From the values obtained by statistical processing. The threshold value may be determined empirically.

  FIG. 1 is a flowchart showing one embodiment of a nucleic acid analysis method. A nucleic acid extraction step, a nucleic acid purification step, a nucleic acid amplification step, and a nucleic acid detection step are performed on the cell sample to be analyzed and the standard cell sample at the same time and under the same conditions. After performing each of the above steps, the detection result is obtained, and the success or failure of all steps of the cell sample to be analyzed is determined based on the success or failure of the nucleic acid analysis result derived from the standard cell sample. Furthermore, the standard deviation is obtained by statistical analysis from the detection results of standard cell samples, and the results of multiple standard cell samples performed simultaneously are compared (simultaneous reproducibility) or compared with the analysis results of another day By performing (day difference reproducibility), the process accuracy of the analysis method can be comprehensively determined.

  FIG. 2 is a flowchart showing another embodiment of the nucleic acid analysis method. A nucleic acid extraction step, a nucleic acid purification step, a nucleic acid amplification step, and a nucleic acid detection step are performed on the cell sample to be analyzed and the standard cell sample at the same time and under the same conditions. The accuracy of each process can be verified by collecting a part of the sample in each process and measuring the mass, concentration, purity, etc. of the nucleic acid in the sample, and obtaining the execution efficiency from these measured values. .

  Such nucleic acid analysis methods can be used for analysis of various nucleic acids such as gene expression level analysis and gene polymorphism analysis, as with other nucleic acid analysis methods. Especially, it can use suitably for the expression level analysis of a gene.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
<Preparation of standard cell sample>
A standard cell sample was prepared using the ORF region of the human c-Myc gene as a standard nucleic acid and a high concentration sodium chloride solution as an inactivation liquid.
First, a recombinant vector (c-Myc expression in which the ORF region of the human c-Myc gene is inserted into the insert region of the vector in which the SD sequence of the expression vector (pET19b, T7 promoter) has been deleted and the protein is not expressed. Vector) was constructed. This c-Myc expression vector was introduced into E. coli BL21 strain for transformation. The obtained transformed Escherichia coli was cultured and induced for expression for 8 hours at 37 ° C. in an automatic expression medium (Overnight Expression Automation System 1, manufactured by Merck) to synthesize mRNA derived from the ORF region of the human c-Myc gene. It was. After expression induction, the transformed Escherichia coli was immersed in a sodium chloride solution (final concentration after mixing with Escherichia coli was 50% (wt / wt)), and an expression profile was immobilized, which was used as a standard cell sample. The standard cell sample was mixed and homogenized, and then subdivided and stored at -20 ° C for 1 day. The standard cell sample after storage for 1 day was in the state of a dispersion dispersed in a sodium chloride solution.

<Nucleic acid analysis method>
Extraction and purification of RNA, reverse transcription reaction using the extracted RNA as a template, nucleic acid amplification reaction using the synthesized cDNA as a template, and detection of the obtained amplification product were performed using the standard cell sample prepared above 60 ng / 100 μl and fecal samples collected from 10 individuals for colorectal cancer screening were simultaneously performed under the same conditions. As the stool sample, stool collected from a human was immersed in a sodium chloride solution (final concentration after mixing with stool was 50% (wt / wt)) and stored at -20 ° C for 1 day. In addition, the standard cell sample was independently tried with respect to three of the subdivided ones.
First, each specimen was centrifuged at 1000 rpm for 5 minutes, the liquid component (sodium chloride solution) was removed with an aspirator, and solids containing cells were collected. To this solid content, 5 ml each of ISOGEN (manufactured by Nippon Gene Co., Ltd.) was added as an extract, and the mixture was stirred with a vortex mixer at 5000 rpm for 30 seconds and mixed well. The obtained mixture was centrifuged at 1000 rpm for 15 minutes, and 4 ml of the supernatant was transferred to another new tube, which was used as an RNA extract. To this RNA extract, 2 ml of chloroform was added as a purified solution, and the mixture was again stirred with a vortex mixer at 5000 rpm for 30 seconds. Thereafter, the mixture was further centrifuged at 1000 rpm for 15 minutes, 1 ml of the aqueous layer was transferred to another new tube, and this was used as an RNA purification solution.

A portion (100 μl) of the RNA extract and RNA purified solution of each specimen was collected, and the 260 nm / 280 nm absorbance ratio and 260 nm / 230 nm absorbance ratio were measured with a spectrophotometer (manufactured by Hitachi High-Technologies Corporation). As a result, all the specimens showed around 2.0 in the 260 nm / 280 nm absorbance ratio, and 1.8 to 1.9 in the 260 nm / 230 nm absorbance ratio. Further, from the absorbance at 260 nm, the RNA concentration of each RNA extract and RNA purified solution was measured, and the purification efficiency was calculated. The purification efficiency of the RNA extract means the ratio of the RNA mass contained in the RNA extract to the RNA mass contained in the sample. Further, the purification efficiency of the RNA purified solution means the ratio of the RNA mass contained in the RNA purified solution to the RNA mass contained in the RNA extract.
As a result, it was confirmed that each sample had an RNA concentration of 0.4 ng / μl or more, a purification efficiency of 80% or more, and a sufficient amount of RNA was extracted and purified. Table 2 shows the 260 nm / 280 nm absorbance ratio, 260 nm / 230 nm absorbance ratio, and RNA purity of the RNA extract and RNA purified solution of the standard cell sample and stool sample calculated from the measurement results. Table 3 shows the RNA concentration, RNA mass, and purification efficiency of the RNA extract and RNA purified solution of the standard cell sample and stool sample. As a result, RNA was extracted and purified well from standard cell samples, and RNA was extracted and purified from all stool samples as well as standard cell samples. It was confirmed that the accuracy was guaranteed. So we went to the next step.

A part of the RNA extract and RNA purified solution of each specimen was collected, and a reverse transcription reaction was performed using a reverse transcription reaction solution (manufactured by Takara Bio Inc., M-MLV). After this reverse transcription reaction, a part of the solution was fractionated, and PCR reaction was performed using a PCR amplification solution (Applied Biosystems, Taq Gold master mix). As a primer for PCR, c-Myc-A primer (SEQ ID NO: 1) and c-Myc-B primer (SEQ ID NO: 2) were used.
Note that the simultaneous reproducibility (N = 3) was examined by statistically processing the results obtained from each of the three subdivided standard cell samples. In addition, this series of steps from nucleic acid extraction to PCR reaction from three standard cell samples stored in small portions was performed once a day for 3 days, and the daily difference reproducibility (N = 27) was examined. Average DNA concentration, standard deviation (SD), and coefficient of variation (CV (%)) in the reaction solution after reverse transcription reaction and nucleic acid amplification reaction using RNA derived from standard cell samples or stool samples
Is shown in Table 4. As a result, it was confirmed that both the standard deviation and coefficient of variation of three trials performed independently at the same time and the standard deviation and coefficient of variation of 27 trials with different implementation dates were small in value and small in variation. It was. From these results, it is clear that the standard cell sample of the present invention is excellent in simultaneous reproducibility and daily reproducibility in a series of steps of nucleic acid analysis.

A part of each reaction solution after the reverse transcription reaction and after the PCR reaction was taken, and amplification products were detected using an electrophoresis apparatus (manufactured by Agilent, Bioanalyzer 2100). From the amount of amplification product and the amount of unreacted primer (residual primer), reverse transcription reaction efficiency and amplification efficiency were calculated. Further, the 260 nm / 280 nm absorbance ratio and 260 nm / 230 nm absorbance ratio of each reaction solution were obtained by a spectrophotometer (manufactured by Hitachi High-Technologies Corporation), and the DNA purity was calculated.
As a result of electrophoresis, bands of 16S rRNA and 23S rRNA were found in the reaction solution after the reverse transcription reaction both in the case of using RNA extracted from a standard cell sample and in the case of using RNA extracted from a stool sample. It was confirmed that in the reaction solution after the PCR reaction, an amplification product band in the ORF region of the human c-Myc gene was confirmed. The ratio of the amount of reaction product to the sum of the amount of reaction product in the reaction solution after the reverse transcription reaction and the amount of residual primer [[reaction product] / ([reaction product] + [residual primer])] is 95.7%. It was confirmed that the reaction efficiency of the reverse transcription reaction was 95% or more. The ratio of the amount of amplification product to the sum of the amount of amplification product and the amount of residual primer in the reaction solution after PCR reaction [[amplification product] / (amplification product] + [residual primer])] is 97.4%. The amplification efficiency of the PCR reaction was also confirmed to be 95% or more. Furthermore, the DNA purity of both reaction solutions after the reverse transcription reaction and after the PCR reaction was 95% or more.
From these results when RNA extracted from a standard cell sample was used, it was confirmed that the process was appropriately performed and the accuracy was guaranteed.

[Example 2]
<Preparation of standard cell sample>
A standard cell sample was prepared using the ORF region of the human COX-2 gene as a standard nucleic acid and an ethanol solution as an inactivation liquid.
First, human COX-2 is inserted into the insert region of a vector in which the SD sequence (the 409th to 413th nucleotide sequences AGGAG) of the expression vector (pET19b, T7 promoter) has been deleted and the foreign protein cannot be expressed. A recombinant vector (COX-2 expression vector) in which the ORF region of the gene was inserted was constructed. This COX-2 expression vector was introduced into Escherichia coli BL21 strain for transformation. The obtained transformed Escherichia coli was cultured for a certain period of time, then IPTG was added as an expression inducer, and expression was induced at 37 ° C. for 2 hours to synthesize mRNA derived from the ORF region of the human COX-2 gene. After expression induction, the transformed Escherichia coli was immersed in ethanol solutions of various concentrations (final concentration after mixing with Escherichia coli was 10, 20, 30, 40, 50, 60, or 70%) to prepare cell samples. These cell samples were mixed and homogenized and then subdivided, and then stored at 4 ° C. for 3 hours or 1 month.

<Nucleic acid analysis using standard cell samples>
Extraction and purification of RNA, reverse transcription reaction using extracted RNA as a template, nucleic acid amplification reaction using synthesized cDNA as a template, and detection of the resulting amplification product for each cell sample prepared above And went respectively.
First, a phenol mixture “Trizol” (manufactured by Invitrogen) was added to each of three subdivided cell samples, and after sufficient dissolution, chloroform was added and mixed thoroughly using a vortex. . The obtained mixture was centrifuged at 12,000 g at 4 ° C. for 20 minutes.
RNA was recovered from the supernatant (aqueous phase) obtained by the centrifugation by an ethanol precipitation method. Specifically, sodium acetate and 100% ethanol were added to the supernatant and stirred, followed by centrifugation to obtain a precipitate, which was washed and air-dried. These precipitates were dissolved in DEPC-treated water to obtain an RNA solution.
1.0 μl of RNA recovered per reaction system was added and reverse transcription was performed to obtain cDNA. Using the obtained cDNA as a template, Taqman PCR was performed using a COX-2 Taqman probe (Hs00153133_m1, Applied Biosystems), and the obtained amplification product was detected. Specifically, 1 μl of each cDNA was dispensed into a 0.2 ml 96-well PCR plate. Thereafter, 8 μl of ultrapure water and 10 μl of nucleic acid amplification reagent “TaqMan GeneExpression Master Mix” (Applied Biosystems) were added to each well, and 1 μl of probe was added and mixed, and the PCR reaction solution was mixed. Prepared. The PCR plate was placed in an ABI real-time PCR apparatus, treated at 95 ° C. for 10 minutes, and then subjected to 40 cycles of thermal cycling at 95 ° C. for 1 minute, 56.5 ° C. for 1 minute, and 72 ° C. for 1 minute. Further, PCR was performed while measuring the fluorescence intensity over time by treating at 72 ° C. for 7 minutes. The measurement result of the fluorescence intensity was analyzed, and the detected COX-2 gene expression level (copy number) was calculated. The calculation results are shown in Table 5 and FIG.

  As a result, when cell samples having an ethanol content of 10% and 20% in a mixture of an ethanol solution and transformed E. coli are used, the expression level is lower than when the ethanol content is 30% to 70%. The variation was also large. Furthermore, after 1 month storage at 4 ° C., the expression level was clearly lower than that after 3 hours storage. On the other hand, when a cell sample having an ethanol content of 30% to 70% was used, it was possible to obtain a stable result of 12000 to 13000 copies and a CV (%) of 3%. In addition, even after 1 month storage at 4 ° C., the expression level was almost the same as that after 3 hours storage. From these results, it is clear that the expression profile in the cells is fixed by immersing the transformed cells in an ethanol solution of 30% or more. In addition, even if the standard cell sample soaked in the inactivation liquid is stored for a long time, the expression pattern of the human-derived gene incorporated in the cell does not change, so the expression level is always Is clearly a stable standard sample.

[Example 3]
<Preparation of standard cell sample>
A standard cell sample was prepared using the ORF region of the human COX-2 gene as a standard nucleic acid and a high salt concentration aqueous solution containing a chelating agent as an inactivation liquid.
First, the COX-2 expression vector used in Example 2 was introduced into Escherichia coli BL21 strain for transformation. The obtained transformed Escherichia coli was cultured for a certain period of time, then IPTG was added as an expression inducer, and expression was induced at 37 ° C. for 2 hours to synthesize mRNA derived from the ORF region of the human COX-2 gene. After expression induction, the transformed Escherichia coli was immersed in an EDTA-containing ammonium sulfate solution (a dispersion after mixing with E. coli was 30 mM EDTA, 30% ammonium sulfate) to prepare a standard cell sample. The standard cell samples were mixed and homogenized, and then divided into three parts, and then each of the three standard cell samples was divided into three patterns under seven temperature conditions of −80 ° C., −20 ° C., 0 ° C., 4 ° C., 20 ° C., 40 ° C., and 60 ° C. Stored for 3 hours, 3 months, 6 months, 9 months, and 12 months.

<Nucleic acid analysis using standard cell samples>
From each standard cell sample after storage, RNA was recovered in the same manner as in Example 2, a reverse transcription reaction was performed using the recovered RNA as a template, and the obtained cDNA was used as a template to obtain a COX-2 Taqman probe (Hs00153133_m1, Taqman PCR was performed using Applied Biosystems. The measurement result of the fluorescence intensity was analyzed, and the detected amplification product amount (COX-2 gene expression level, copy number) was calculated. The calculation results are shown in Tables 6 to 8 and FIG.

  As a result, when stored at 60 ° C., the expression level rapidly decreased depending on the storage period. In contrast, when stored at 40 ° C. or lower, the expression level did not change until 6 months. In particular, when stored at 20 ° C. or lower, the expression level did not change even when stored for 12 months. From these results, it is clear that the standard cell sample immersed in the inactivating liquid can be stably stored for a long time at room temperature.

  A nucleic acid having a series of steps of extraction and purification of nucleic acid from a cell sample, amplification of the extracted nucleic acid, and detection of an amplification product by the method for producing a standard cell sample of the present invention, as well as excellent storage stability. Since a standard cell sample suitable as a standard sample for analysis accuracy control can be easily produced, it is particularly useful in the field of nucleic acid analysis, particularly in the field of clinical examination for medical diagnosis.

Claims (17)

  Inactivation of a transformed cell into which an expression vector incorporating a standard nucleic acid with a known base sequence in whole or in part is introduced, while suppressing variation in the expression profile while retaining the nucleic acid inside the cell A method for producing a standard cell sample, comprising an inactivation treatment step of immersing in a liquid.   The standard cell sample according to claim 1, wherein mRNA synthesized using the standard nucleic acid as a template is present in the transformed cell, but a protein derived from the standard nucleic acid is not produced. Manufacturing method. Before the inactivation process step,
Contacting an expression inducer to synthesize mRNA in the transformed cell using the standard nucleic acid as a template;
The method for producing a standard cell sample according to claim 1 or 2, wherein:   The production of a standard cell sample according to any one of claims 1 to 3, wherein RNA transcribed from the region containing the standard nucleic acid in the expression vector is deficient in ribosome binding ability. Method.   The said inactivating liquid is a solution which uses as an active ingredient 1 or more types selected from the group which consists of a water-soluble organic solvent, a protease inhibitor, a chelating agent, and salts. A method for producing a standard cell sample according to claim 1.   The method for producing a standard cell sample according to claim 5, wherein the water-soluble organic solvent is at least one selected from the group consisting of water-soluble alcohols, ketones, and aldehydes.   The standard cell according to claim 5 or 6, wherein the inactivating liquid contains a water-soluble alcohol and / or ketone as a water-soluble organic solvent, and the concentration of the water-soluble organic solvent is 30% or more. Sample manufacturing method.   The method for producing a standard cell sample according to claim 6 or 7, wherein the water-soluble alcohol is at least one selected from the group consisting of ethanol, propanol, and methanol.   The method for producing a standard cell sample according to any one of claims 6 to 8, wherein the ketones are acetone and / or methyl ethyl ketone.   The standard cell sample according to claim 5 or 6, wherein the inactivating liquid contains an aldehyde as a water-soluble organic solvent, and the concentration of the water-soluble organic solvent is 0.01 to 30%. Method.   The standard cell sample according to any one of claims 5 to 10, wherein the inactivating liquid contains sodium chloride at a concentration of 13% (wt / wt) or more to a saturation concentration or less. Method.   The method for producing a standard cell sample according to any one of claims 5 to 11, wherein the inactivating liquid contains ammonium sulfate at a concentration of 30% (wt / wt) or more to a saturation concentration or less. .   The method for producing a standard cell sample according to any one of claims 5 to 12, wherein the inactivating liquid contains 0.1 mM to 1 M chelating agent. After the inactivation treatment step,
A storage step of storing the transformed cells in a dispersion in an inactivated liquid at −20 to 40 ° C .;
The method for producing a standard cell sample according to any one of claims 1 to 13, wherein   The method for producing a standard cell sample according to any one of claims 1 to 14, wherein the standard nucleic acid is a nucleic acid derived from a human gene.   16. The method for producing a standard cell sample according to claim 1, wherein the standard nucleic acid is a COX-2 (cyclooxygenase-2) gene-derived nucleic acid or a c-Myc gene-derived nucleic acid.   The method for producing a standard cell sample according to any one of claims 1 to 16, wherein the transformed cell is a prokaryotic cell.

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