JP2009276204A - Method, device, and program for analysis of gene expression - Google Patents

Abstract

The present invention proposes a gene expression analysis method, a gene expression analysis apparatus, and a program capable of improving accuracy.
For each of a plurality of nucleic acid probes, data indicating a complementary strand formation amount with a target nucleic acid extracted from a target cell is obtained, and for each of the plurality of acquired data, each complementary strand formation amount indicated by the data is obtained. The component amount of adenine, guanine, cytosine and thymine in the total amount is calculated, and a reference value for comparison between the data is determined from the calculated component amount of each base.
[Selection] Figure 13

Description

  The present invention belongs to a technical field related to normalization, analysis or correction of gene expression levels obtained using a bioassay platform such as a DNA chip.

  Conventionally, the amount of complementary strand formation between a plurality of designed DNA probes and cDNA obtained by converting mRNA extracted from a sample cell with reverse transcriptase was measured by fluorescence intensity, and the measurement result was expressed in the sample cell. There is a technique for detecting the gene expression level.

  This gene expression level varies depending on external factors such as external stress on the sample cell to be extracted and differences in conditions or skill when extracting mRNA from the sample cell. Therefore, for example, when investigating a difference in gene expression level per unit time for sample cells in the same tissue, or when investigating a difference in gene expression level for sample cells in different tissues, etc. When comparing the data indicating the gene expression levels obtained, it is important to normalize the gene expression levels between the data because the external factors are not the same.

As a standardization technique, the number of repetitive sequences present in a DNA sample strongly correlates with the amount of hybridization, but the repetitive sequences are present in the genome at a substantially constant rate, so that the repetitive sequences present in the DNA sample are present. Focusing on the fact that the sequence strongly correlates with the number of cells, using the number of cells as an index, the obtained gene expression level is converted to a value per unit cell number, so that it can be compared with other gene expression levels. Have been proposed (see, for example, Patent Document 1).
JP 2006-170670 A

  However, in such a standardization method, a control for obtaining the number of cells must be fixed to the DNA chip. Therefore, in this standardization method, the control itself may change due to external factors such as the environment or procedure in the process until the control is fixed, and the reliability of standardization accuracy is poor. Become.

  The present invention has been made in consideration of the above points, and intends to propose a gene expression analysis method, a gene expression analysis apparatus, and a program capable of improving accuracy.

  In order to solve this problem, the present invention provides a gene expression analysis method, an acquisition step for acquiring data indicating the amount of mRNA expressed in a target cell, and adenine in the amount indicated by the data acquired in the acquisition step And a component amount calculation step for calculating at least one component amount among the component amounts of guanine, cytosine, and thymine or uracil, and the component amount calculated for each data in the component amount calculation step, between the data A comparison reference determination step for determining a reference value for comparison is performed.

  Further, the present invention is a gene expression analyzer, an acquisition unit for acquiring data indicating the amount of mRNA expressed in a target cell, adenine in the amount indicated by the data acquired by the acquisition unit, guanine, A component amount calculation unit that calculates at least one of the component amounts of cytosine and thymine or uracil, and a reference value for comparison between the data from the component amounts calculated for each data by the component amount calculation unit It is set as the structure containing the comparison reference | standard determination part to determine.

  Moreover, this invention is a program, Comprising: Acquiring the data which shows the quantity of mRNA expressed by a target cell with respect to an acquisition part, The data acquired by the acquisition part with respect to a component amount calculation part show Calculate at least one component amount among the component amounts of adenine, guanine, cytosine, thymine or uracil in the amount, and the component amount calculation unit calculates for each data with respect to the comparison reference determination The determination of the reference value for comparison between the data is performed from the amount of the component.

  In general, it is known that cells try to keep various substances such as the amount of ATP (adenosine triphosphate) in the inside (homeostasis). From this, even if the gene expression level of each gene expressed in the cell changes, the total amount of the gene expression level is considered to be approximately constant, and this has already been confirmed by the present applicant. . Therefore, the total amount of base components in the amount of mRNA expressed in the target cell serves as an index value for obtaining a comparison standard between the data to be compared.

  In the present invention, since the total amount of base components in the amount of mRNA expressed in the target cells is obtained by calculation, a reference for comparison between data even without a control for obtaining an index value or a reference value The value can be determined, and as a result, changes in the reference value due to the control itself can be eliminated. Thus, it is possible to realize a gene expression analysis method, a gene expression analysis apparatus, and a program that can improve accuracy.

  Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings.

(1) Overall Configuration of Measurement System FIG. 1 shows the overall configuration of the measurement system 1 according to this embodiment. The measurement system 1 includes a nucleic acid chip 2x (x = 1, 2,..., Or j (j is an integer)), a fluorescence intensity reader 3 and a gene expression analyzer 4. The nucleic acid chip 2x, the fluorescence intensity reader 3 and the gene expression analyzer 4 will be described in order.

(2) Nucleic acid chip The nucleic acid chip 2x is a base on which nucleic acid probes corresponding to all or part of genes contained in target cells such as eukaryotes or prokaryotes are arranged. A nucleic acid probe is a single-stranded nucleotide that functions as a probe (detector) for detecting a specific gene.

  In general, a nucleic acid probe is not a nucleotide having a base sequence that interacts with an entire specific gene, but is designed as a plurality of probe fragments (hereinafter also referred to as a probe set) that contain a base sequence that is specific to the gene. Is done. Specifically, it is designed as a DNA (deoxyribonucleic acid) fragment, cDNA (complementary DNA) fragment or PNA (peptide nucleic acid) of about 18 to 60 [mer].

  In the case of this embodiment, on the arrangement surface of the nucleic acid chip 2x, for example, as shown in FIG. 2, k regions (k is an integer of 2 or more) corresponding to the number of genes to be measured (in the figure, A portion surrounded by a broken line) is assigned, and for each region, one probe set and a control for each probe fragment in the probe set (hereinafter referred to as a fragment control) are arranged in x rows and y columns. The

  In the nucleic acid chip 2x, the target nucleic acid extracted from the target cell is given to the probe set in each region, and a complementary strand formation reaction between the nucleic acid probe (probe set) and the target nucleic acid (hereinafter referred to as this). Also referred to as hybridization). In general, mRNA or pre-mRNA or a fragment thereof is not used as the target nucleic acid, but one obtained by converting the mRNA or pre-mRNA or a fragment thereof with a reverse transcriptase is used.

In the case of this embodiment, in order to measure the transition of the gene expression level in the target cell, the target nucleic acid is extracted from the passaged target cell at a predetermined time point, and the target nucleic acid at each time point corresponds to the corresponding nucleic acid chip 2x _1. Is given to each probe set at 2 ₂ ,. Further, a labeling substance is added to the target nucleic acid before and after hybridization. The labeling substance is generally a fluorescent dye such as biotin or FITC (fluorescein isothiocyanate), but is not limited thereto, and may be, for example, a radioisotope.

(3) Fluorescence intensity reading device The fluorescence intensity reading device 3 (FIG. 1) is arranged on the arrangement surface of the nucleic acid chip 2x arranged at a predetermined reading position when a reading request is given from the gene expression analysis device 4. Irradiate the excitation light of the labeling substance added to the target nucleic acid. When the nucleic acid probe and the target nucleic acid form a complementary strand, the labeling substance added to the target nucleic acid emits light by excitation light. The amount of luminescence correlates with the amount of complementary strand formed with the target nucleic acid, and the amount of luminescence increases as the amount of complementary strand formed between the target nucleic acid and the nucleic acid probe increases.

  The fluorescence intensity reading device 3 reads the amount of light emitted from the probe fragment and the fragment control in units of probe sets arranged on the nucleic acid chip 2x, and the read light amount data (hereinafter referred to as fluorescence intensity data). Is output to the gene expression analyzer 4.

(4) Gene Expression Analyzer (4-1) Circuit Configuration of Gene Expression Analyzer As shown in FIG. 3, the gene expression analyzer 4 is a CPU (Central Processing Unit) that controls the entire gene expression analyzer 4 10, a ROM (Read Only Memory) 11 in which a program for executing gene expression analysis processing (hereinafter also referred to as a gene expression analysis program) is stored, and a RAM (Random Access as a work memory of the CPU) Memory) 12, operation unit 13, storage unit 14, interface 15, and display unit 16 are connected to each other via a bus 17.

  When the gene expression analysis program stored in the ROM 11 is expanded in the RAM 12, the CPU 10 appropriately controls the storage unit 14, the interface 15 and the display unit 16 based on the gene expression analysis program, and executes the gene expression analysis process. Has been made.

(4-2) Processing Contents of CPU Based on Gene Expression Analysis Program The CPU 10 that has expanded the gene expression analysis program in the RAM functionally has a base number acquisition unit 21 and a fluorescence intensity acquisition unit 22 as shown in FIG. , Expression level calculation unit 23, base component amount calculation unit 24, comparison criterion determination unit 25, normalization unit 26, and notification unit 27.

  The base number acquisition unit 21 waits for an input request for the number of bases from the operation unit 13 (FIG. 3). When the input request is received, the base number acquisition unit 21 is arranged on the nucleic acid chip 2x using, for example, the display unit 16 (FIG. 3). For each probe set, it is required that the number of adenine, guanine, cytosine and thymine (hereinafter also referred to as 4 base numbers) of each probe fragment should be input. Incidentally, the conceptual diagram regarding this 4 base number is shown in FIG.

  Further, when data indicating the number of 4 bases of each probe set (hereinafter also referred to as 4 base number data) is input as a response to the input request, the base number acquiring unit 21 stores the 4 base number data in the storage unit 14. (FIG. 3).

  The fluorescence intensity acquisition unit 22 waits for a fluorescence intensity reading request for the nucleic acid chip 2x from the operation unit 13 (FIG. 3). When the reading request is received, the interface 15 (FIG. 3) is used to A reading request is made to the connected fluorescence intensity reading device 3 (FIG. 1).

  Further, when the fluorescence intensity acquisition unit 22 acquires fluorescence intensity data from the fluorescence intensity reader 3 as a response to the reading request, for example, an acquisition date and an acquisition number are used as identifier data (hereinafter referred to as a chip) for the nucleic acid chip 2x. (Referred to as identification data).

  When the fluorescence intensity acquisition unit 22 acquires fluorescence intensity data, the expression level calculation unit 23 calculates a gene expression level for each probe set based on the fluorescence intensity data, and the calculated gene expression level of each probe set (Hereinafter referred to as expression level data) is stored in the storage unit 14 (FIG. 3) in association with chip identification data indicating the acquisition source of the fluorescence intensity data.

  The amount of gene expression is an estimated amount that indicates the gene expressed in the target cell, and the amount of luminescence correlated to the amount of complementary strand formation between the target nucleic acid and nucleic acid probe approaches the amount of true complementary strand formation. Is statistically corrected.

  In the case of this embodiment, the gene expression level is calculated as a ratio of the luminescence level by using version 5 of data analysis software called MAS (Micro Array Suite) of Affymetrix.

  Here, the MAS 5 will be briefly described by paying attention to one probe set. In MAS5, (1) the local physical influence (background) is excluded from the light emission amount of each probe fragment in the probe set. (2) The light emission amount of each probe fragment (referred to as a perfect match probe) is appropriately corrected according to the difference between the probe fragment and the corresponding fragment control (referred to as a mismatch probe). (3) The amount of luminescence of each probe fragment (referred to as a perfect match probe) is calculated as a gene expression level by logarithmic conversion.

  For details, see I.S.Kohane / A.T.Kho / A.J.Butte by Yoshi Hoshida, Microarray Data Analysis for Integrated Genomics, Springer Japan Publishing, p. 58-74.

  The base component amount calculation unit 24 waits for a comparative analysis request for gene expression levels between nucleic acid chips from the operation unit 13 (FIG. 3), and when receiving the comparative analysis request, two or more chip identification data and four bases Whether the numerical data is stored in the storage unit 14 (FIG. 3) is recognized.

Here, when two or more chip identification data do not exist in the storage unit 14, this means that the fluorescence intensity data has not yet been read from the two or more nucleic acid chips 2 ₁ , 2 _2. It means that comparative analysis of gene expression level cannot be performed. In addition, when the 4-base number data does not exist in the storage unit 14, this means that there is no 4-base number data that is essential for obtaining a reference standard for gene expression levels between the expression level data. Means you can't. In these cases, the base component amount calculation unit 24 notifies, for example, via the display unit 16 (FIG. 3) that the gene expression level cannot be compared and analyzed between the expression level data and the cause thereof.

  On the other hand, when both the two or more chip identification data and the 4-base number data exist in the storage unit 14, the base component amount calculation unit 24 includes all the chip identification data stored in the storage unit 14, The expression level data associated with the chip identification data and the 4-base number data are read out, and based on these data, the base component amount in the gene expressed in the target cell is calculated.

  That is, the base component amount calculation unit 24, based on the chip identification data and expression level data read from the storage unit 14, for example, in each probe set for each nucleic acid chip 2x (each elapsed time point) as shown in FIG. In addition to recognizing the gene expression level, the number of adenine, guanine, cytosine and thymine (FIG. 5) in each probe set is recognized based on the 4-base number data.

  Then, as shown in FIG. 7, the base component amount calculation unit 24 multiplies the gene expression level in each probe set by the adenine number, guanine number, cytosine number, and thymine number for each nucleic acid chip 2x. This multiplication result means the amount of each base component in the gene expression level in the probe set.

  Thereafter, as shown in FIG. 8, the base component amount calculation unit 24 adds the multiplication results in units of the same type for each nucleic acid chip 2x. This addition result means the amount of base components in the total gene expression level of each probe set in the nucleic acid chip 2x, that is, the amount of base components of each base in the gene expressed in the target cell.

  As described above, even if the gene expression level of each gene expressed in a cell changes, the total amount of the gene expression level is considered to be approximately constant. This has already been confirmed by the present applicant. It was. Therefore, the addition result (the amount of base components of each base in the gene expressed in the target cell) is an index value for obtaining a reference for comparative analysis of gene expression levels between expression level data (between nucleic acid chips). .

  Here, experimental results are shown in FIGS. FIG. 9 shows the transition of the amount of each base component for a predetermined period of time (nucleic acid chip 2x) while KLF4 (Kruppel Like Factor 4) is continuously applied to the colon cancer cells as a stimulus. Incidentally, as the nucleic acid chip, GDS1942-HG-U133A from Affymetrix was used, and the gene expression level was calculated by MAS5 from Affymetrix.

  FIG. 10 shows the transition of the amount of each base component for a predetermined period of time (nucleic acid chip 2x) by continuously giving epidermal growth factor (EGF) as a stimulus to breast cancer cells. . Incidentally, GDS2324-HG-U133A from Affymetrix is used as the nucleic acid chip, and the gene expression level is calculated by MAS5 from Affymetrix.

  FIG. 11 shows changes in the amount of each base component after a predetermined period of time (nucleic acid chip 2x) when TGFb1 is added to normal ovarian epithelium. Incidentally, GSE6653-HG-133 from Affymetrix was used as the nucleic acid chip, and the gene expression level was calculated by RMA (Robust Multichip Analysis) from Takara Bio.

  As is clear from FIGS. 9 to 11, it can be seen that the component amount of each base is approximately constant regardless of the elapsed time (nucleic acid chip 2x). This is because, as described above, the expression level of each gene expressed in a living cell is different, but the total expression level of each gene expressed in the cell is constant. Therefore, as the number of probe sets arranged on the nucleic acid chip 2x increases, in other words, as the number of probe sets corresponding to the total genes that can be expressed in the target cell approaches, the amount of each base component in the nucleic acid chip 2x becomes the expression level. As an index value for obtaining a reference for comparative analysis of gene expression levels between data (between nucleic acid chips), certainty (reliability) is increased.

  When the amount of each base component occupying the total gene expression level in the nucleic acid chip 2x is calculated, the comparison criterion determination unit 25 performs a comparative analysis of the gene expression level between the expression level data (between the nucleic acid chips) in the same type of base unit. Determine the reference value.

  In this embodiment, as shown in FIG. 12, the reference value for each base is the average of the amount of base components in each nucleic acid chip 2x (each elapsed time).

  When the reference value for each base is calculated, the normalization unit 26 calculates the difference (variation degree) between the base component amount and the reference value in each nucleic acid chip 2x (each elapsed time) in the same type of base unit. When this difference is less than a predetermined threshold value, this means that noise elements other than the gene expression level, such as local physical influence (background), are small.

  The normalization unit 26 determines that the base component amount of each base is constant for each nucleic acid chip having a base component amount whose difference from the reference value for each base is less than a threshold value in each nucleic acid chip 2x (each elapsed time). After being corrected, the gene expression level in each nucleic acid chip 2x (each elapsed time) is displayed on the display unit 16 in a predetermined display mode.

  In this correction, for example, a predetermined statistical method such as correction with a correction amount weighted according to the difference from the reference value is employed.

  The notification unit 27 may have low reliability with respect to a nucleic acid chip having a base component amount that is less than a threshold value at least even one reference value for each base among the nucleic acid chips 2x (each elapsed time). For example, the display unit 16 is notified of both or one of the high and high values to be re-experimented.

  As described above, the CPU 10 that has expanded the gene expression analysis program in the RAM can reduce the nucleic acid if the relative variation in the amount of each base component of each base out of the total gene expression amount in each nucleic acid chip 2x (each elapsed time) is small. Assuming that gene expression levels can be compared between chips, the base component amount of each base is corrected to be constant.

(4-3) Gene Expression Analysis Process Procedure The gene expression analysis process described above is performed according to the flowchart shown in FIG. However, FIG. 13 shows the procedure when 4-base data has already been acquired.

  That is, the CPU 10 starts this measurement process with, for example, power-on as a trigger, and determines whether or not a request for obtaining fluorescence intensity at the nucleic acid chip 2x has been received in step SP1.

  Here, when receiving the fluorescence intensity acquisition request, the CPU 10 proceeds to step SP2 to generate expression amount data indicating the gene expression amount from the fluorescence intensity data given from the fluorescence intensity reader 3, and this is chip-identified. The data is stored in the storage unit 14 in association with the data, and the process returns to step SP1. On the other hand, if the acquisition request for fluorescence intensity has not been received, the CPU 10 proceeds to step SP3 to determine whether or not a request for comparative analysis of the expression level has been received. Return to step SP1.

  On the other hand, if the CPU 10 receives the expression level comparison analysis request, the CPU 10 proceeds to step SP4 and determines whether or not the comparison request analysis precondition is satisfied based on the data stored in the storage unit 14. To do.

  Here, as described above, when both of the two or more chip identification data and the 4-base number data are not present in the storage unit 14, it means that the precondition for the comparison request analysis is not satisfied. In this case, the CPU 10 proceeds to step SP5 to notify that the gene expression level cannot be compared and analyzed between the nucleic acid chips and the cause thereof, and returns to step SP1.

  In this manner, the CPU 10 stores the expression level data of each nucleic acid chip 2x (each elapsed time) in the storage unit 14 until the precondition for the comparison request analysis is satisfied.

  When the preconditions for the comparison request analysis are satisfied, the CPU 10 proceeds to step SP6 and calculates the base component amount of each base in the total gene expression amount for each nucleic acid chip 2x (predetermined elapsed time) (FIG. 7 and FIG. 7). 8), in the subsequent step SP7, the index value of each base is calculated from the calculation result (FIG. 12).

  Further, the CPU 10 proceeds to step SP8 to calculate the degree of variation between the amount of the base component and the index value in each nucleic acid chip 2x (each elapsed time) in the same base unit, and the degree of variation from the index value of each base. If there is at least one nucleic acid chip having a base component amount that is less than or equal to the threshold value, in step and 9, it is notified that the nucleic acid chip should be re-experimented, and the process proceeds to step SP10.

  Then, in step SP10, the CPU 10 corrects the base component amount of each base so that the base component amount of each base is constant for the nucleic acid chip having the base component amount whose variation from the index value of each base is less than the threshold value. Based on the result, the gene expression level in each nucleic acid chip 2x (each elapsed time) is displayed on the display unit 16 in a predetermined display mode, and then the measurement processing procedure is terminated.

  In this manner, the CPU 10 can execute the measurement processing procedure and display the nucleic acid chip whose gene expression level can be compared on the display unit 16 in a predetermined display mode.

(5) Operation and Effect In the above configuration, the gene expression analyzer 4 acquires a plurality of expression level data indicating the gene expression level with the target nucleic acid for each of the plurality of nucleic acid probes. Then, the gene expression analyzer 4 calculates the amount of components of adenine, guanine, cytosine, and thymine in the total amount of each gene expression level indicated by the expression level data for each expression level data (FIGS. 6 to 8). A reference value for comparison between the expression level data is determined from the calculated component amount (FIG. 12).

  The amount of base components in the total amount of gene expression indicated by the expression level data is the expression level because the total amount of gene expression level is roughly constant due to the fact that the substances present in the cells are kept constant. This is an index value for obtaining a standard for comparative analysis of gene expression levels between data (between nucleic acid chips).

  Since the gene expression analyzer 4 calculates the amount of the base component in the total amount of gene expression indicated by the expression level data, even if there is no control for obtaining the index value, comparison between the expression level data is possible. The reference value can be determined.

  Usually, the control serves as an index for the nucleic acid chip 2x, and does not serve as an index between the nucleic acid chips. Therefore, when comparing the expression level data obtained in different laboratories, it is not known whether the difference is due to different conditions in each laboratory. In this regard, the gene expression analyzer 4 can determine a reference value for comparison between expression level data without the control for obtaining the index value. This is particularly useful when comparing quantitative data.

  Here, in the expression level data, the amount of complementary strand formation with a target nucleic acid for a plurality of nucleic acid probes is read as a physical quantity (in this embodiment, the amount of luminescence) by a sensor, and the physical quantity is converted by a statistical method. It is a thing.

  Since this statistical method eliminates background (local physical effects), etc., the data used as the basis for calculating the amount of base components is less than in the case of data indicating physical quantities read by sensors. High reliability. Therefore, the gene expression analysis apparatus 4 uses a reference value for comparison between expression level data as a condition for extracting external stress on a target cell or mRNA from the sample cell, as compared with data indicating a physical quantity. Alternatively, it can be determined as close to a true value without external factors such as a difference in skill. However, it is possible to determine the reference value for comparison between the expression level data, even if the data that is the basis for calculating the amount of the base component is data indicating the physical quantity instead of the expression level data, and it is possible to determine the usefulness described above. There is.

  In addition, since the base component amount occupies the total amount of each nucleic acid probe and the complementary strand formation amount with the target nucleic acid, the larger the number of nucleic acid probes (the number of nucleic acid probes arranged on the nucleic acid chip 2x ( The higher the number of probe sets), the better. On the other hand, as described above, the total sum of gene expression levels correlated with the amount of ATP existing in cells is constant. Therefore, if the number of nucleic acid probes is the number corresponding to the total gene that can be expressed in the target cell, the reliability of the amount of base component in the total amount of gene expression indicated by the expression level data is the highest.

  In addition, when there is a component amount in which the difference from the reference value is greater than or equal to a threshold value among the component amounts of each base, the gene expression analyzer 4 re-experiments the expression amount data from which the component amount is calculated. Notify what to do. Therefore, this gene expression analysis apparatus 4 uses external factors such as external stress on the target cell and differences in conditions or skill when extracting mRNA from the sample cell among a plurality of expression level data to be compared. Since it is possible to identify and present which expression level data is large, it is possible to avoid redoing all experiments. In addition, when comparing the expression level data obtained in different laboratories, it is possible to specify the laboratory that obtained the expression level data with a large external factor. As a result, it is possible to improve the experimental accuracy between laboratories.

  Moreover, this gene expression analyzer 4 correct | amends so that the component quantity from which the difference with a reference value is less than a threshold value among the component quantities of each base may become fixed. Therefore, the gene expression analyzer 4 can eliminate errors due to procedures and environmental factors related to the measurement except for extremely bad measurement results.

  According to the above configuration, a plurality of expression level data indicating a gene expression level with a target nucleic acid is acquired for each of a plurality of nucleic acid probes, and for each acquired expression level data, each gene expression level indicated by the expression level data is obtained. By calculating the amount of components of adenine, guanine, cytosine, and thymine in the total amount, and by determining the reference value for comparison between the expression amount data from these component amounts, even if there is no control, between the expression amount data Since it is possible to determine the reference value for comparison in the above, it is possible to eliminate the change in the reference value caused by the control itself, and thus to realize the gene expression banquet apparatus 4 that can improve the accuracy.

(6) Other Embodiments As an acquisition mode for acquiring data indicating the amount of mRNA expressed in the target cell, in the above-described embodiment, the gene expression level of the target nucleic acid in the cell is determined using the nucleic acid chip 2. The expression level data shown was acquired. However, the acquisition mode is not limited to this embodiment. For example, an embodiment in which each mRNA expressed in the target cell is extracted and directly obtained by proliferating to a certain amount using real-time PCR (Polymerase Chain Reaction) is also applicable.

  In addition, for example, a mode in which data indicating the amount of mRNA is acquired from a data storage medium is also applicable. When this aspect is applied, for example, it is possible to calculate an average index value at the experimental location using data indicating the amount of mRNA obtained at a plurality of remote experimental locations.

  Examples of the data storage medium include package media such as a flexible disk, CD-ROM (Compact Disk-Read Only Memory), and DVD (Digital Versatile Disc), a semiconductor memory in which data is temporarily or permanently stored, There are magnetic disks. In addition, as a method for acquiring data from these data storage media, a wired or wireless communication medium such as a local area network, the Internet, or digital satellite broadcasting can be used.

  Further, as the reading amount, in the above-described embodiment, the light emission amount is optically read. However, the reading amount is not limited to this embodiment. For example, an electromagnetic quantity or an impedance quantity can be applied electromagnetically. In short, a reading amount read by a sensor that reads a predetermined physical quantity can be applied. As the nucleic acid chip 2x, for example, a Stanford type manufactured by Affymetorix can be applied, and other than these can also be applied.

  In addition, the reading place is the nucleic acid chip 2x in the above-described embodiment. However, the reading place is not limited to this. For example, a tissue section can be applied and other locations can also be applied.

  Further, MAS is applied as a method for calculating the gene expression level in the above-described embodiment. However, the calculation method is not limited to this. For example, known data analysis software such as RMA or other new calculation methods can be applied. In short, it is sufficient if data indicating the amount of complementary strand formation read from the sensor is corrected using a statistical method.

  In addition, as a method for calculating the amount of base component, in the above-described embodiment, adenine, guanine, cytosine, and thymine (occupying uracil when the nucleic acid probe is mRNA) occupying the formation amount of the nucleic acid probe and the target nucleic acid. Each component amount was calculated. However, it is only necessary to calculate at least one of these component amounts.

  Incidentally, for example, when calculating the component amount of only adenine, normalization of bases other than adenine is obtained by multiplying the base component amount of guanine by the guanine number / adenine number, and the cytosine number with respect to the base component amount of cytosine. / Adenine number multiplied is the correction target. Also, the amount of base component of thymine (uracil) multiplied by the number of thymine (uracil) / adenine is to be corrected. In this way, the same effect as in the case of the above-described embodiment can be obtained. But you may make it correct | amend by methods other than this correction | amendment.

  In addition, as a method for calculating the index value, in the above-described embodiment, the average amount of base components in each nucleic acid chip 2x (each elapsed time) is used. However, the index value calculation method is not limited to this embodiment. For example, the maximum value, the minimum value, or the intermediate value of the base component amount in each nucleic acid chip 2x (each elapsed time) can be applied.

  In addition, the calculation target of the index value is the same cell in the above-described embodiment, but it may be a heterogeneous cell. In order to calculate the index value in a heterogeneous cell, the component amount of each base occupying the amount of target nucleic acid and nucleic acid probe formed in the heterologous cell may be multiplied by the ratio of the total number of genes between the different cells. The total number of genes between different types of cells can be obtained, for example, by uploading the association between the cell species and the total number of genes in the species to the server for each species (type) of the nucleic acid chip, and obtaining from the server. .

  In addition, as a method for acquiring the number of four bases of each probe set, the one input from the operation unit 13 is acquired in the above embodiment. However, the acquisition method is not limited to this embodiment. For example, a combination of a probe set arranged on a nucleic acid chip and the number of four bases in the probe set is uploaded to a server for each type (type) of nucleic acid chip, and wired or wireless communication is performed from the server. It is possible to apply a mode of selecting and acquiring using a medium.

  The present invention can be used in the bio-industry such as genetic experiments, creation of medicines, or patient follow-up.

It is a basic diagram which shows the whole structure of the measurement system by this Embodiment. It is a basic diagram with which it uses for description of fixation of a probe set and a control. It is a block diagram which shows the circuit structure of a gene expression analyzer. It is a block diagram which shows the functional structure of CPU at the time of gene expression analysis processing mode. It is a basic diagram with which it uses for description of 4 base number in a probe set. It is a basic diagram with which it uses for description of the expression level of the probe set in each nucleic acid chip. It is a basic diagram with which it uses for description of calculation of the amount of base components in a probe set. It is a basic diagram with which it uses for description of calculation of the amount of base components in each nucleic acid chip. It is a graph which shows an experimental result (1). It is a graph which shows an experimental result (2). It is a graph which shows an experimental result (3). It is a basic diagram with which it uses for description of calculation of the reference value with respect to each base. It is a flowchart which shows a gene expression analysis processing procedure.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Measuring system, 2x ... Nucleic acid chip, 3 ... Fluorescence intensity reader, 4 ... Gene expression analyzer, 10 ... CPU, 11 ... ROM, 12 ... RAM, 13 ... Operation part, 14 ...... Storage unit, 15 ... Interface, 16 ... Display unit, 21 ... Base number acquisition unit, 22 ... Fluorescence intensity acquisition unit, 23 ... Expression amount calculation unit, 24 ... Base component amount calculation unit, 25 …… Comparison criteria determination unit, 26 …… Normalization unit, 27 …… Notification unit.

Claims (11)

An acquisition step of acquiring data indicating the amount of mRNA expressed in the target cell;
A component amount calculating step for calculating at least one component amount among the component amounts of adenine, guanine, cytosine, and thymine or uracil in the amount indicated by the data acquired in the acquisition step;
A gene expression analysis method comprising: a comparison reference determination step for determining a reference value for comparison between data from the component amount calculated for each data in the component amount calculation step. In the above acquisition step,
For each of the plurality of nucleic acid probes, obtain data indicating the amount of complementary strand formation with the target nucleic acid extracted from the target cell,
In the component amount calculating step,
For each of a plurality of data acquired in the acquisition step, at least one component amount of the component amounts of adenine, guanine, cytosine, and thymine or uracil occupying the total amount of each complementary strand formation indicated by the data The gene expression analysis method according to claim 1, wherein An acquisition unit for acquiring data indicating the amount of mRNA expressed in the target cell;
A component amount calculation unit that calculates at least one component amount among the component amounts of adenine, guanine, cytosine, and thymine or uracil in the amount indicated by the data acquired by the acquisition unit;
A gene expression analysis apparatus comprising: a comparison reference determination unit that determines a reference value for comparison between data from the component amount calculated for each data by the component amount calculation unit. The acquisition unit
For each of the plurality of nucleic acid probes, obtain data indicating the amount of complementary strand formation with the target nucleic acid extracted from the target cell,
The comparison criterion determination unit
For each of a plurality of data acquired by the acquisition unit, at least one component amount of the component amounts of adenine, guanine, cytosine, and thymine or uracil occupying the total amount of each complementary strand formation indicated by the data The gene expression analysis apparatus according to claim 3, wherein   The gene expression analysis apparatus according to claim 4, wherein the plurality of nucleic acid probes have probes corresponding to all genes that can be expressed in a target cell. Among the component amounts calculated for each piece of data by the component amount calculation unit, when there is a component amount whose difference from the reference value determined by the comparison reference determination unit is equal to or greater than a threshold, the calculation source of the component amount The gene expression analysis apparatus according to claim 4, further comprising a notification unit that notifies that data should be re-experimented. The component amount calculation unit
A base number acquisition unit that acquires at least one base number among the base numbers of adenine, guanine, cytosine, and thymine or uracil in the plurality of nucleic acid probes;
The arithmetic unit that multiplies each of the plurality of data by each complementary strand formation amount indicated by the data and the number of bases acquired by the base number acquisition unit, and adds each multiplication value. Gene expression analyzer. A normalization unit that corrects the component amount calculated for each piece of data by the component amount calculation unit so that the component amount whose difference from the reference value determined by the comparison reference determination unit is less than a threshold value is constant. The gene expression analyzer according to claim 4. The above data is
For each of a plurality of nucleic acid probes, the amount of complementary strand formation with the target nucleic acid is read by a sensor, and the read physical quantity is statistically converted so as to approach the amount of complementary strand formation. The gene expression analysis apparatus according to claim 4. Acquiring the data indicating the amount of mRNA expressed in the target cell to the acquisition unit,
Calculating at least one component amount among the component amounts of adenine, guanine, cytosine, and thymine or uracil in the amount indicated by the data acquired by the acquisition unit for the component amount calculation unit;
A program for executing a determination of a reference value for comparison between data from a component amount calculated for each piece of data by the component amount calculation unit with respect to a comparison reference determination part. For the acquisition unit, for each of a plurality of nucleic acid probes, acquiring data indicating the amount of complementary strand formation with the target nucleic acid extracted from the target cell,
A component of adenine, guanine, cytosine, and thymine or uracil occupying the total amount of complementary strand formation indicated by the data for each of a plurality of data acquired by the acquisition unit with respect to the component amount calculation unit The program according to claim 10, wherein at least one component amount among the amounts is calculated.

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