JP3880361B2 - Fluorescence signal processing method and hybridization reaction result display method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a biochip capable of precisely observing the characteristics of a plurality of types of biopolymers such as DNA contained in a sample, and a method for evaluating the accuracy of detection results.
[0002]
[Prior art]
As the number of species whose genome sequence has been determined increases, genes that appear to correspond to evolution are discovered, and a set of genes that all organisms are thought to have in common is searched. In order to make a guess, so-called genome comparison methods that try to find something from the genetic differences between species have been actively performed. However, in recent years, with the development of infrastructure represented by DNA microarrays and DNA chips (hereinafter collectively referred to as biochips), the interest of molecular biology has shifted from information between species to information within species, that is, simultaneous expression analysis. Along with comparisons between species so far, the field of association from the extraction of information has begun to spread greatly.
[0003]
For example, if an unknown gene showing the same expression pattern as a known gene is found, it can be inferred that the gene has the same function as the known gene. The functional meaning of these genes and proteins themselves has been studied in the form of functional units and functional groups. In addition, the interaction between them can be caused by destroying or overreacting a gene by matching with known enzyme reaction data and substance metabolism data, or more directly, or eliminating the gene expression It is expressed and the direct and indirect effects of the gene are analyzed by examining the expression pattern of all genes.
[0004]
When examining a gene expression pattern, in an experiment using a biochip, first, an element related to a biological tissue to be examined is prepared. Here, the element is a fragment of DNA related to the biological tissue to be tested in a single-stranded state. This element is placed on a substrate such as a glass slide or silicon, and a plurality of the same type of elements are arranged. In summary, biochips are spotted and fixed at a density of several hundred to several thousand per square centimeter. This collection of spotted elements is called a spot. FIG. 15 shows the relationship between the element 1502 and the spot 1501 on the biochip 1500. FIG. 15 also shows an enlarged schematic diagram of one spot 1501. The target is a DNA or RNA obtained by fragmenting DNA extracted from a biological tissue to be tested in a single-stranded state, and refers to a substance that reacts with an element on a biochip. When a gene in the cell is expressed, the DNA is transcribed into RNA. This RNA is extracted and fluorescently labeled as a target. When the target and the element are reacted, single strands in a complementary relationship are bound to each other and hybridized. In the biochip, the expression state of the gene in the living tissue can be observed using hybridization.
[0005]
A successful example in this area is the report of experimental results on the efficacy of drugs by Tsunoda et al. (T. Tsunoda et al .: Discrimination of Drug Sensitivity of Cancer Using cDNA Microarray and Multivariate Statistical Analysis: Genome Informatics 1999 (1999, Dec.) pp.227-228, Universal Academy Press Inc.). The process of this experiment is shown in FIG. In the experiment, RNA extracted from normal cells and RNA extracted from cancer cells are labeled with fluorescent dyes of different colors, mixed in equal amounts, and hybridized with an element (gene) on a biochip, The intensity of the fluorescent signal generated from both fluorescent dyes is measured.
[0006]
FIG. 2 is a diagram schematically showing a method for displaying the expression state of each gene obtained by this experiment. This display is a plot of the fluorescence signal data of the normal cell-derived target and the cancer cell-derived target hybridized with the gene on the biochip, with one axis (Y axis) representing the normal cell and the other axis ( The X axis shows the fluorescence signal of cancer cells. One plot in the figure corresponds to one gene. In the data analysis, in this XY plane, the area on the right side of the straight line X = k or the upper side of the straight line Y = k, and the area between the Y axis and the straight line Y = mX, or the X axis and Y = (1 / M) Focus on the area between X. The former is to eliminate minute fluorescent signals due to noise, the latter is a gene that works in normal cells but does not work in cancer cells, or conversely a gene that works in cancer cells but does not work in normal cells Is for finding. m is usually selected from 2 to several tens depending on the degree of significance for cancer cells or normal cells. Therefore, the genes that fall into the region A and the genes that fall into the region B in FIG. By adopting such a display method, it is possible to narrow down gene candidates that specifically act on a specific disease.
[0007]
[Problems to be solved by the invention]
As the nature of the biochip, the intensity of the fluorescent signal obtained from the experiment does not necessarily correspond to the amount of RNA contained in the actual sample. For example, when the fluorescence signal of spot A on the biochip observes twice the fluorescence signal of spot B, the amount of the gene corresponding to spot A and the gene corresponding to spot B contained in the sample is doubled. Not necessarily. This is because the amount of spotting on the biochip is not constant, the amount of elements present in the spot is not uniform, the spot viscosity is not constant, the binding force between the element and the target varies from element to element, temperature and humidity The technology has not been able to cope with environmental changes such as, the technology for maintaining a constant hybridization reaction efficiency on the biochip, and the reading accuracy of fluorescence after hybridization have been fully established experimentally. This is because there are various factors such as not.
[0008]
Therefore, usually, a researcher normalizes the obtained fluorescence signal data and pays attention to a qualitative portion of the data. In other words, when observing the expression status of each gene in normal cells and cancer cells, which element (gene) was specific, or when observing the change in the expression of each gene during the cell division cycle, From the fluorescence signal data, we read which elements (genes) tend to become stronger in expression from late to late. Therefore, biochip experiments must be reproducible in a qualitative sense. However, in an actual experiment, the range of the fluorescence signal from each spot is large, and the intensity of the signal cannot be measured accurately when the fluorescence signal is read as image data and digitized. An example will be described below.
[0009]
First of all, a fluorescent signal is emitted very strongly from the spot because of the large amount of target contained in the sample or the number of elements contained in the spot, which can be read when digitizing the scanned image. If you exceed the upper limit of the numerical value, you may not be able to measure. In this case, the measured value of the fluorescence signal is usually replaced with the upper limit of the readable value. FIG. 3 is a diagram illustrating a case where the behavior of a gene is measured between two samples as shown in FIG. 1, but a spot exceeding the reading limit is measured. A spot 301 surrounded by a dotted circle in FIG. 3 is a spot exceeding the reading limit. Although this spot 301 was strongly expressed in a sample of cancer cells, it was observed beyond the reading limit, so it was replaced by the maximum value of the signal. If the reading limit range is wider, this spot may have been included in region B. As described above, since the intensely emitted fluorescence signal could not be used effectively, there was a risk of missing useful information.
[0010]
Conversely, since the fluorescence signal is small, there is a risk that useful information may be missed. A rectangular region surrounded by a dotted circle 401 in FIG. 4 is a portion that is not a target of data analysis because it is a small fluorescent signal. This is because it cannot be determined whether the fluorescent signal from the spot is derived from noise or whether it has been observed to be really small. There are important genes that induce the function of other genes even with small expression, so we want to avoid missing such information. In view of the characteristics of such a biochip and a fluorescence measuring instrument and the nature of data obtained from the fluorescence measuring instrument, the present invention provides a biochip that is effective for acquiring as much information as possible from an experiment using a biochip and The object is to provide a data analysis method.
[0011]
[Means for Solving the Problems]
In order to achieve the above object, in the present invention, a plurality of elements having different element concentrations are spotted on one chip. Here, the difference in element density means a difference in the amount of elements contained in the spot. This is schematically shown in FIG. If the spot 1601 contains twice as many elements as the elements contained in the spot 1602, it is assumed that the spot 1601 has a density twice that of the spot 1602.
[0012]
For example, as shown in FIG. 5, five types with concentrations of 20%, 40%, 60%, 80%, and 100% are prepared. Here, all the spots having a density of 100% may not have the same amount of elements. The density may be determined to be 20%, 40%, 60%, and 80% relative to the spot, assuming that the element amount of the spot for each element type is 100%.
[0013]
FIG. 6 shows an example in which fluorescence signals are read from this biochip and plotted on a plane. At this time, when the reading limit is exceeded with a high density spot such as spot D1 or spot E1 for element 1, it is a thin spot with a density that does not exceed the reading limit, such as spot A1, spot B1, or spot C1. Data analysis may be performed. On the other hand, if the fluorescence signal is weak and the data is not subject to data analysis, such as spot A2, spot B2, and spot C2, with respect to element 2, the object of data such as spot D2 and spot E2 Data analysis may be performed with a dark spot that does not enter the outside. In addition, when the spot of all concentrations exceeds the reading limit, such as spot A3, spot B3, spot C3, spot D3, and spot E3 for element 3, on the contrary, the fluorescence signal is emitted at spots of all concentrations. If it is too small to be included in the area that is not subject to data analysis, these fluorescent signal data should not be used for data analysis. In such a case, the number of types of density may be further increased so as to find a spot that enters the target region for data analysis. In this way, by preparing spots of a plurality of concentrations for the same element, it is possible to avoid missing a useful gene that may have been missed in the past.
[0014]
That is, the present invention is characterized in that, in a biochip in which spots of a plurality of types of elements are arranged and arranged on a substrate, a plurality of spots having different element concentrations are arranged for each element. The plurality of spots containing the same element do not necessarily have to be arranged spatially in one place on the substrate. It suffices if it is known at which position on the substrate which element spot of which density is arranged.
[0015]
The fluorescence signal processing method according to the present invention uses a substrate on which a plurality of types of elements are arranged with a plurality of spots having different element concentrations for each element, and is arranged on a fluorescently labeled target contained in a sample and the substrate. Performing a hybridization reaction with the elements of the spot, detecting the fluorescent signal emitted from each spot on the substrate, digitizing the detected fluorescent signal intensity, and determining the concentration of the element containing the same element. Of the fluorescence signals detected from different spots, data analysis is performed by excluding the fluorescence signal whose digitized signal intensity shows the maximum value of digitization and the fluorescence signal whose signal intensity is smaller than a preset threshold level. And a step.
[0016]
In the fluorescence signal processing method according to the present invention, a plurality of types of elements are labeled with different fluorescent dyes derived from two types of specimens using a substrate on which a plurality of spots having different element concentrations are arranged for each element. A step of performing a hybridization reaction between a target and an element of a spot arranged on the substrate, a step of detecting two kinds of fluorescent signals emitted from each spot on the substrate, and digitizing the detected fluorescent signal intensity Of the fluorescent signals detected from multiple spots with the same element and different element concentrations, the digitized signal intensity shows the maximum value of digitization and the signal intensity is smaller than a preset threshold level Same as excluding fluorescent signal For a plurality of spots with different element concentrations including mentament, the ratio of the two types of fluorescence signals detected for each spot was calculated, the ratio average and variance were calculated, and the ratio average and variance were preset. Comparing the value with each other to determine whether the experiment is successful for each element.
[0017]
The present invention also provides a target in which a plurality of types of elements are labeled with a substrate on which a plurality of spots having different element concentrations are arranged for each element, and with different fluorescent dyes derived from the first specimen and the second specimen. In the method of displaying the results of the hybridization reaction between a plurality of types of elements, a target derived from the first sample, and a target derived from the second sample performed using a sample containing On the two-dimensional coordinates where the target derived from one specimen is the fluorescent signal intensity emitted from the labeled fluorescent dye, and the other axis is the fluorescent signal intensity emitted from the fluorescent dye labeled from the target derived from the second specimen A step of displaying a plurality of marks corresponding to each spot and designating one of the marks displayed on the two-dimensional coordinates A method, characterized by comprising the step of highlighting all the marks relating to the same elements as the specified mark.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, an embodiment for carrying out the present invention will be specifically described with reference to the drawings.
FIG. 7 is a block diagram showing a configuration example of a biochip system that performs biochip production, fluorescence signal detection, and signal data analysis.
This biochip system includes a central processing unit 700 for inputting spot information and analyzing experimental data, a display unit 701 for displaying characters and graphic screens, and a keyboard 702 for inputting values to the system and for selecting operations. And a mouse 703, a biochip information database 704 in which spot elements, position information on the biochip, signal measurement values, and the like are stored. The central processing unit 700 includes an element placement unit 710 that reads from the biochip information database and instructs the spotter to place the element at a predetermined position, and a signal analysis unit 711 that analyzes the read fluorescence signal. It is embodied by the program.
[0019]
The element placed in the well 705 is taken out by the spotter 706 and spotted at a predetermined position on the biochip 707. The element on the biochip 707 is hybridized with the target in the sample by the hybridization experimental apparatus 708, and the fluorescence signal from the spot of the biochip after the hybridization is read by the detector 709. The fluorescence signal read by the detector 709 is input to the central processing unit 700 and analyzed by the signal analysis unit 711.
[0020]
FIG. 8 is a diagram showing an example of element data managed by this system, and is stored in the biochip information database 704. Element data is stored in an array of eNum structures of length element [i] (i = 1, 2,..., ENum). Where eNum is the total number of element types stored in this database. The array element [] has an element ID 800 for uniquely identifying the element in the database, a sequence name 801, a DNA sequence 802, a sequence length 803 of the DNA sequence, and an annotation 804 in which the nature of this sequence is written. Consists of. In addition, information such as the name of the biological tissue (organ) from which the sequence is extracted, the name of the organism, the EST, the accession number of GENBANK, the primer sequence, and the like may be added as attributes of element [].
[0021]
FIG. 9 is a diagram showing an example of chip information data managed by this system, and is stored in the biochip information database 704. The biochip information data is stored in an array of cNum structures having a length of chip [i] (i = 1, 2,..., CNum). However, cNum is the total number of target biochips stored in this database. The array chip [] is an experimental environment information 903 in which a chip ID 900 that uniquely determines a biochip in this database, a chip name 901, an experiment date 902, experimental equipment used in this biochip experiment, temperature setting, and the like are described. , Information 904 of the sample used in this biochip. In addition to this, various information on the biochip may be added as the attribute of chip [].
[0022]
FIG. 10 is a diagram illustrating an example of spot data managed by the present system, and is stored in the biochip information database 704. The spot data is stored in an array of sNum structures having a length of spot [i] (i = 1, 2,..., SNum). However, sNum is the total number of spots on the target biochip stored in this database. The array spot [] is a chip ID 1000 indicating which chip is located on a chip, a coordinate position 1001 on the chip, an element ID 1002 indicating what element this spot is, and what percentage of the spot is from the original dilution. Dilution degree 1003 indicating whether it was diluted by a ratio (a spot having an element of the element contained in the original dilution) 1003, and signal 1 indicating the intensity of the fluorescent signal obtained from this spot (1004) , Signal 2 (1005), signal 1 to signal 2 ratio 1006, average ratio 1007 between spots having the same element, and dispersion 1008. In this structure, an experiment between two samples is assumed, but in the case of an experiment with a single sample, only signal 1 should be used, and if comparison with three or more samples is assumed. Just increase the number of members related to signal strength.
[0023]
FIG. 11 is a flowchart showing an outline of biochip production according to the present invention and analysis processing of fluorescence signal data obtained from the biochip.
First, the element placement unit 710 reads element data, chip information data, and spot data from the biochip information database 704, and instructs the spotter 706 to spot the element designated at a predetermined position. The spotter 706 sucks the element from the well 705 and places it on the biochip 707 (step 1100). When spotting spots with different concentrations (dilution degrees), those with different concentrations may be prepared in the well 705 in advance, or once spotted, the chip is dried to evaporate the water in the spots. There is also a method of increasing the density by repeating spotting at the same position from the beginning.
[0024]
The produced biochip is hybridized with the sample by the hybridization experiment apparatus 708 (step 1101). After hybridization, the fluorescence signal from the probe on the chip is read by the detector 709 (step 1102). Finally, only the data included in the data analysis target range is identified, the signal ratio and the like are calculated (step 1103), and the processing is terminated by displaying the result (step 1104).
[0025]
FIG. 12 is a detailed flow of the processing of step 1103 in which the signal data in FIG. 11 is analyzed, only the data included in the data analysis target range is identified, and the signal ratio and the like are calculated.
First, the signal intensity of each spot is calculated from the signal (image data) read by the detector 709 in step 1102, and the spot where the signal is too strong and exceeds the reading limit, that is, the fluorescence signal data is read. The spot corresponding to the signal replaced at the limit is invalidated (step 1200).
[0026]
Next, normalization is performed on signal data of spots other than the invalidated spot. There are two ways to normalize, and if one looks at two very close samples (such as changes in the same cell over time), the fluorescence signal of many spots will not change, In this method, the sum of the fluorescence signals from the spots is regarded as a reference value of the biochip signal, and the relative value therefrom is used as the signal intensity of each spot. For example, when the fluorescence signals are measured as 100, 200, and 500 from the fluorescence signals in the cancer cells of three spots A, B, and C, respectively, the fluorescence signal in the cancer cells of the spot A after normalization is 0.125 (= 100 / (100 + 200 + 500)), the fluorescence signal in spot B cancer cells is 0.25, and the fluorescence signal in spot C cancer cells is 0.625. Similarly, normalized values are obtained from fluorescent signals in normal cells of spots A, B, and C.
[0027]
Another way of thinking is that when using samples that are considered to be significantly different in expression, this should be corrected by using a positive control that is considered to have a certain amount of expression in all samples such as housekeeping genes. This is considered to be a standard for signal 0, and is considered to be a standard for signal 0 using those that are considered to be hardly expressed in all samples (negative control), such as genes that are expressed only in plants for animal experiments. This is a method in which the signal intensity of each spot is determined by a relative value from the reference. For example, fluorescence signals are measured as 100, 200, and 500 from the fluorescence signals in cancer cells at three spots A, B, and C, respectively, and 1000 and 50 from the fluorescence signals in cancer cells of the positive control gene and the negative control gene, respectively. , The fluorescence signal in the cancer cell of the spot A after normalization is 0.053 (≈ (100-50) / (1000-50)), the fluorescence signal in the cancer cell of the spot B is 0.158, The fluorescence signal in the cancer cells in spot C is 0.474. Similarly, normalized values are obtained from fluorescent signals in normal cells of spots A, B, and C.
[0028]
If the normalized value is a decimal point and it is difficult to handle in calculation, there is a method of multiplying it by a constant to make it an integer value. The fluorescence signal in the cancer cell after normalization and the fluorescence signal in the normal cell are registered in signal 1 (1004) and signal 2 (1005) of the spot data (step 1201).
Next, the ratio of signal 1 to signal 2 is obtained for the signal data of all normalized spots. That is, "signal 2 value / signal 1 value" is obtained and registered in the spot data ratio 1006 (step 1202).
[0029]
Further, the average and variance of the ratio between spots having the same element and different concentrations (array spot [] having the same chip ID and element ID) are calculated. However, a spot where the signal data shows a small value may be a signal due to noise, and is excluded from the calculation target. That is, when both signal 1 (1004) and signal 2 (1005) are both below a certain threshold value (k), they are excluded from calculation.
[0030]
For example, the spots A1, B1, C1, D1, and E1 are spots having the same element and different densities. The normalized values of the fluorescence signal in cancer cells of these spots and the fluorescence signal in normal cells are (63, 51) for spot A1, (201, 110) for spot B1, (359, 195), it is assumed that the spot D1 is (449, 219) and the spot E1 is (631, 302). Further, a threshold value k regarded as a noise signal is set to 100.
[0031]
Then, the ratio of each spot calculated in step 1202 is as follows: spot A1 is 0.81 (≈51 / 63), spot B1 is 0.55, spot C1 is 0.54, spot D1 is 0.49, spot E1 Is 0.48. However, since the two signal values (63 and 51) of the spot A1 are both lower than the threshold value k, the ratio of spots other than the spot A1 is calculated when calculating the average / dispersion of the ratio. Then, the average is 0.51 (≈ (0.55 + 0.54 + 0.48 + 0.47) / 4), and the dispersion is 0.001 (≈ {(0.55-0.51). ² + (0.54-0.51) ² + (0.48-0.51) ² + (0.47-0.51) ² } / 4).
[0032]
The average / dispersion in the element thus obtained is registered in the average 1007 of the ratio of each spot data and the variance 1008 of the ratio. An element with a large dispersion value indicates that the spot has impurities or that the hybridization reaction has failed. If a certain threshold value is set and the variance value exceeds this value, it is determined that the experiment is not successful (step 1203).
[0033]
FIG. 13 shows an example of a result display screen of the present system, in which signals corresponding to spots are plotted (1301) on the XY plane 1300 based on normalized data. On the plane, threshold values X = k and Y = k for determining a range for eliminating a minute fluorescence signal due to noise, and threshold values Y = mX and Y = (1) indicating which sample is significant. / M) A straight line of X is drawn so that useful gene information is easy to see. Further, when a point plotted with the mouse 703 is selected, spot points with different densities having the same element on the plane are highlighted, and the average of these ratios and the value of the variance of the ratios are displayed in a balloon 1306. The The smaller the variance, the better the spot is in the experiment. In another window, the chip name, experiment date and time, sample information, the name of the element selected with the mouse, annotations, etc. can be referenced as biochip information.
[0034]
FIG. 14 shows another example of the result display screen of the present system, in which fluorescent signal data obtained from each spot is displayed in a table. The table is composed of the element name, the dilution from the original dilution, the signal of sample 1, the signal of sample 2, the average and variance of the signal ratio, and the characteristics of the sample. Compare the mean and variance of each element's ratio with a preset value to determine which sample it is significantly inclined to, or whether the experiment is successful, and automatically enter it into the feature column By filling in, you can easily understand the nature of the element.
Through the above processing, it becomes possible to observe the characteristics of a plurality of types of biopolymers such as DNA contained in a sample more precisely than before.
[0035]
【The invention's effect】
According to the present invention, by preparing spots with a plurality of concentrations, it is possible to observe spot data that could not be observed due to the fluorescence signal being too weak or too strong, which may have been missed in the past. Never miss a gene that has a useful function.
[Brief description of the drawings]
FIG. 1 is an explanatory diagram of a method of using a biochip for comparing gene expression states between two samples.
2 is an explanatory diagram of a display example of an experimental result obtained in the experiment of FIG. 1. FIG.
FIG. 3 is an explanatory diagram when there is a spot that exceeds the reading limit of the fluorescence signal.
FIG. 4 is an explanatory diagram showing a region where the fluorescence signal is too small to be used for data analysis.
FIG. 5 is an explanatory diagram of an example of a biochip according to the present invention.
FIG. 6 is an explanatory diagram of a display example of an experimental result obtained from the biochip of this method.
FIG. 7 is a block diagram showing a configuration of a biochip system according to the present invention.
FIG. 8 is a diagram showing an example of the data structure of element data.
FIG. 9 is a diagram showing an example of the data structure of chip information data.
FIG. 10 is a diagram showing an example of the data structure of spot data.
FIG. 11 is a flowchart showing an outline of biochip production according to the present invention and analysis processing of fluorescence signal data obtained from the biochip.
FIG. 12 is a flowchart showing details of fluorescence signal data analysis.
FIG. 13 is an explanatory diagram showing an example of an experiment result display of this system.
FIG. 14 is an explanatory diagram showing another example of the experiment result display of this system.
FIG. 15 is a diagram showing the relationship between elements and spots.
FIG. 16 is an explanatory diagram of element density.
[Explanation of symbols]
301: Spot exceeding the reading limit 401: Area where the signal from the spot is small and is not regarded as an object of data analysis, 700: Central processing unit, 701 ... Display device, 702 ... Keyboard, 703 ... Mouse, 704 ... Biochip information Database, 705 ... Well, 706 ... Spotter, 707 ... Biochip, 708 ... Hybrid experiment apparatus, 709 ... Detector, 710 ... Element placement unit, 711 ... Signal analysis unit, 1500 ... Biochip, 1501 ... Spot, 1502 ... Element

Claims (3)

Hybridization of fluorescently labeled targets contained in a sample and spot elements arranged on the substrate using a substrate on which a plurality of elements are arranged with a plurality of spots having different element concentrations for each element Performing a reaction;
Detecting a fluorescent signal emitted from each spot on the substrate;
Digitizing the detected fluorescence signal intensity;
Among the fluorescent signals detected from multiple spots with the same element and different element concentrations, the fluorescent signal whose digitized signal intensity shows the maximum value of digitization and the fluorescent signal whose signal intensity is smaller than the preset threshold level And a step of performing data analysis by excluding the fluorescence signal processing method. Using a substrate on which a plurality of types of elements are arranged with a plurality of spots having different element concentrations for each element, targets labeled with different fluorescent dyes derived from two types of specimens and spots arranged on the substrate Performing a hybridization reaction with the element;
Detecting two types of fluorescent signals emitted from each spot on the substrate;
Digitizing the detected fluorescence signal intensity;
Among the fluorescent signals detected from multiple spots with the same element and different element concentrations, the fluorescent signal whose digitized signal intensity shows the maximum value of digitization and the fluorescent signal whose signal intensity is smaller than the preset threshold level Step to exclude,
Calculating a ratio of the two kinds of fluorescence signals detected for each spot for a plurality of spots having different element concentrations containing the same element, and calculating an average and variance of the ratios;
Fluorescent signal processing method characterized by comprising the steps you value and compared to a preset the dispersion prior SL min. A plurality of types of elements, a substrate on which a plurality of spots having different element concentrations are arranged for each element, and a sample containing targets labeled with different fluorescent dyes derived from the first specimen and the second specimen In the method of displaying the results of the hybridization reaction between the plurality of types of elements and the target derived from the first sample and the target derived from the second sample,
One axis is the fluorescent signal intensity emitted from the fluorescent dye labeled with the target derived from the first specimen, and the other axis is the fluorescent signal intensity emitted from the fluorescent dye labeled with the target derived from the second specimen. Displaying a plurality of marks corresponding to each spot on the two-dimensional coordinates,
Designating one of the marks displayed on the two-dimensional coordinates;
And a step of highlighting all marks relating to the same element as the designated mark.

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