1. Field of the Invention
The present invention relates to a display method of measurement information such as a measured object, a measurement condition, an image of a detection result, numerical data and a comparison result by detecting a biochemical reaction of a solid-phase support with solid-phased probes to detect a biologically relevant material.
2. Description of Related Art
A microarray has been known as an example of a solid phase support of which a probe to detect a biologically relevant material is solid-phased. For example, DNA probes which are spotted on the microarray are mixed with a fluorescently labeled nucleic acid fluid originating from a cell to measure the amount of nucleic acid extracted from a cell to be compared, thereby detecting the intensity of fluorescence. As the amount of the nucleic acid is measured by using the intensity of fluorescence, it is possible to examine the expression level of a gene, the presence of a particular gene in a genome, and the gene mutation.
Thus, it is critical to measure the amount ratio of nucleic acid and display a measurement result without difficulty. The appropriate display of the measurement result may improve precision in analyzing the measurement result with the microarray and save time in analyzing the measurement result. An example of the display means includes, as disclosed in Japanese Unexamined Patent Application, First Publication (JP-A) No. 2001-41892, a three dimensional graph in which an axis X refers to a set of test samples, an axis Y is a microarray set and an axis Z presents the intensity of fluorescence.
The invention provides a display method of measurement information of a biologically relevant material which measures the amount of biologically relevant material of a test sample or the amount ratio of the biologically relevant material among a plurality of test samples, by using a microarray, and displays the measurement information on a computer screen.
In the invention, “biologically relevant material” includes various materials which exist or originate from a living body (animal cells, plant cells and microorganism cells, and viruses that proliferate only if being parasitic on those cells), and includes natural, artificially composite materials (e.g. genetically artificial), etc. For example, the biologically relevant material may include nucleic acids such as DNA, cDNA and RNA, proteins such as several types of hormones, a tumor marker, an enzyme, an antibody and an antigen; and complexes such as RNA.
In the invention, “shade” refers to color gradation and color intensity of a carrier or a medium, and may be recognized as differences of optical properties. The difference may be recognized by both a device and the human eye.
“Measurement information” includes overall information such as bibliographic facts including a measured object, a position of a probe spot a serial number of a microarray, name of experimenter, experiment data and place, a type of a test sample; a measurement condition including a measuring device, a measurement method, a type of an optical filter, a measurement temperature, pH measurement time; a measurement result such as signal intensity data; and an analysis result of the measurement result such as a data normalization method, the calculation of an amount of the biologically relevant material and an amount ratio of the biologically relevant material among the plurality of test samples.
The display method of the measurement information of the biologically relevant material according to the present invention may have the following aspects (1) to (5).
(1) A replicate image which illustrates a layout of a probe spot in the microarray is displayed as the measurement information.
(2) The amount of the biologically relevant material of the test sample which is measured by using the microarray is normalized to compare the amount of the biologically relevant material in the each test sample.
(3) A bar graph illustrates the amount ratio of the biologically relevant material of two test samples to be compared with each other.
(4) The relationship between the position of the probe spot and the amount ratio or signal intensity of the biologically relevant material among the two test samples to be compared, with respect to the plurality of probe spots on the micro array, is displayed with a polygonal line graph or a plot.
(5) An image which is a combination of images generated under optimal signal detecting conditions for the each probe spot is displayed.
It is possible to select the display method of the data, e.g. displaying the result individually or overlapping and displaying the result. For example, if two test samples are compared with each other, the signal intensity of a sample 1 is displayed in a first gradation, e.g. in red color while the signal intensity of a sample 2 is displayed in a second gradation, e.g. in green color. Then, the overlapped signal intensity is displayed in a gradation mixed with the first and second gradations. As the test samples have different gradations, the data may be analyzed easily by varying the gradations with respect to the signal intensity or the ratio in a probe spot layout.
The data may be analyzed by varying shades with respect to the amount of the biologically relevant material. For example, a low expression level of a gene is displayed in green, a high expression level of a gene is displayed in red and an intermediate expression level of a gene is displayed in yellow. Thus, the overall probe spots are represented in 256 gradations, thereby enabling to analyze the amount and the ratio of the biologically relevant material of the respective probe spots at a glance.
If the signal intensity of the probe spot oh the replicate image gradually changes from the center to the circumference, it is similar to a genuine probe spot. That is, the image of each replicate probe spot is displayed on the replicate image in a circle whose color density is higher at the center and gradually becomes lower toward the circumference thereof. Thus, a user may easily get the image as the probe spot layout. If the position of the probe spot on the microarray is displayed on the replicate image, a user may check the signal intensity of the probe spots depending on positions and check whether the amount ratio of the biologically relevant material changes by viewing only the overall layout.
It is possible to select one of raw signal intensity data and normalized data (normalized data) which removes unbiochemical noises therefrom, as the displayed data depending on the analyzed content. For example, the foregoing data may be used to visually compare the overall differences of the raw data or compare the amount of the biologically relevant material. It is preferable to display the signal intensity raw data and the normalized data by interchanging them.
It is more preferable that the detailed information on each probe spots is displayed. Such an information display method includes a method of displaying a mark on the probe spots of the replicate image. As shown in
It is preferred that information on the used and unused probe spots may be adjusted on the replicate image. For example, as shown in
According to another display method, as shown in
The example of the display method of the replicate image is described above. The display method or the information display items are not limited to those mentioned above, and may vary as long as they are appropriate to analyze the microarray.
When the amount of the biologically relevant material is compared by using test samples obtained from a cell to be compared to analyze the microarray data, unbiochemical noise or bias such as the difference of the RNA amount included in the respective test samples occur in the microarray data. The noises or the bias may be caused by the different fabrications of the test samples. However, it is technically difficult to fabricate test samples not to cause noise or the bias. Thus, it is required to normalize the data (normalization of the bias of the data) between the microarrays from the obtained signal intensity data.
The example of normalization includes a method that the data may be normalized by using an internal control gene if the biologically relevant material is a gene. The internal control gene refers to a gene which does not change with respect to any test sample, e.g. in expression. Thus, if two types of test samples are compared and if the signal intensity value of the internal control gene in the sample 1 is 100 and the signal intensity value of the internal control gene in the sample 2 is 200, the result of the sample 1 can double to normalize the noise or the bias due to the differences in the RNA amount.
As it is important to mark the position (probe spot) of the internal control gene in the analysis result in the course of analysis, the position of the internal control gene may be preferably marked in any part of the analysis result.
Even if the internal control gene is selected as the unchanged gene at a design stage, it may not be appropriate to use the unchanged gene to normalize the data due to experiment failures. If the selected gene is not the internal control gene in the analysis result, it should be reset. Meanwhile, a gene which was not the internal control gene at the design stage may be reset as the internal control gene. As shown in
Another normalization method of the analysis result includes a method of using an average signal intensity value or an intermediate value of the overall probe spots. This method is employed on the assumption that the reaction of the genes arranged on the microarray is not different between the test samples. Thus, the overall detectable genes on the microarray are used to normalize the analysis result. The present method is used if it is difficult to determine the internal control gene.
Three normalization methods are described above. A user may select the normalization method while analyzing the microarray data. Preferably, the three normalization methods are used alternately. The current result needs to be changed as a new value to be displayed. If the normalization method is changed, it is important to know which normalization method is used for the currently-displayed result. As shown in
There are provided three normalization methods above. However, the normalization method is not limited to the three methods described above, and may vary as long it is appropriate to normalize the microarray data. The display and setting methods which are illustrated with the drawings are not limited to those described above, and may vary as long as they can provide and adjust information.
To analyze the microarray data, the compared data, the conditions and the type of the test samples used in the experiment should be considered.
For example, the displayed measurement information may include a folder storing the measurement information having the analysis result, the folder name, the type of the test samples, a serial number of the microarray, the name of the experimenter, the type of an exciting and absorbing optical filter used to measure the biologically relevant material, a measurement condition such as the measurement date and place, a measurement temperature, pH and measurement time, the amount of the biologically relevant material and the amount ratio of the biologically relevant material among the plurality of test samples. For example, if the folder storing the measurement information having the analysis result or the folder name is displayed, a user can acknowledge the storing place of the respective data such as the displayed analysis result. Even if a user forgets which data is used, the user can determine the used data by examining the relationship between the original data and the analysis result. Likewise, as the type of the test samples is displayed, a user may recognize the type of the test samples used to obtain the respective analysis data. Therefore, a user can determine whether the type of test samples is correctly used in analysis.
The displayed measurement information is not limited to those described above, and may vary as long as it is necessary to analyze the biologically relevant material including the microarray with respect to various items simultaneously.
To analyze a microarray, the amount of a biologically relevant material is compared by using test samples obtained from a cell to be compared. As shown in
To visualize the comparison result more easily, the bar graph may have shades. For example, the shades of the bar graphs may vary depending on the amount ratio of the biologically relevant material. More specifically, 0.5 and below refers to the declined amount ratio of nucleic acid and the corresponding bar graph is displayed in a first shade, e.g. red color. Arrange which is larger than 0.5 and smaller than 2 represents the unchanged amount ratio of nucleic acid, and the corresponding bar graph is displayed in a second shade, e.g. yellow, while 2 and above refers to the raised amount ratio of nucleic acid and the corresponding bar graph spot is displayed in a third shade, e.g. green color. The different shades are used to identify the amount ratio of nucleic acid so that a user may recognize the result more easily. If the currently-analyzed probe spot is displayed in a fourth shade, e.g. blue, a user may recognize the displayed result more easily. Instead of the method of varying the colors of the bar graphs displaying the various amount ratios of nucleic acid, a method of illustrating the bar graph thicker with respect to the high expression level of the gene may be used. The thickness of the bar graph may vary or the bar graph may be marked with, e.g. an asterisk and the comparison result may be displayed with the different number of the marks.
The present method can be used in the replicate image in
The display state of the bar graph may depend on the type of the biologically relevant material. Thus, the information on the biologically relevant material is sequentially arranged in a vertical or horizontal axis. If the biologically relevant material is a gene, the display sequence may include a gene sequence and an amount ratio sequence of nucleic acid, etc. Preferably, the display sequence is determined according to analysis content. As shown in
The range of the amount ratio of the biologically relevant material (in this case the amount of nucleic acid) displayed in the horizontal or vertical axis may be preferably changed. In some cases, a user may desire to check a limited range in more detail depending on the type of analysis even though the data range obtained is not determined. In such a case, as shown in
The test samples which are selected as a reference (e.g. results from a normal cell) to be compared are changed in the experiment. Preferably, the test samples are selected, referring to the data. For example, if the amount of nucleic acid between two test samples, i.e., between Sample 1 and Sample 2 is compared, Sample 1/Sample 2 or Sample 2/Sample 1 can be calculated and displayed. As shown in
If the measurement information is arranged as shown in
According to the foregoing examples, the display state is changed or checked by clicking the right button of the mouse on the bar graph to display the window, but not limited thereto. Alternatively, a user may adjust or check the display state from a menu bar or a tool bar.
The display method of the analysis result of the microarray may include a method of displaying a change in the amount of nucleic acid over all probe spots.
In the graph of the amount ratio of the biologically relevant material and the signal intensity data, it is possible to extract a case where extremely-strange normalization was carried out if a user can select whether to use the raw data or the normalized data.
If a user uses a selection unit e.g. places a mouse pointer on the plot to display a window including position information of a genomic DNA fragment in a chromosome, a gene name, a probe name and numerical data, he/she can receive detailed information on the plot from the graph.
A high signal or a very low signal may be detected from probe spots on a microarray according to reacted test samples. For example, if the expression levels of a gene are analyzed, the probe spot at a high signal intensity level represents a gene at a high expression level while the probe spot at a low signal intensity level represents a gene at a low expression level. As the signal intensity of the respective probe spots on the microarray is compared the values of genes in both high and low expression levels should be calculated precisely. Even if the probe spots at both high and low signal intensity levels are present on the microarray, the respective probe spots may be provided with the optimal exposure conditions. An image of the respective probe spots obtained under the optimal exposure conditions should be displayed.
Hereinafter, the signal detecting method will be described with reference to
An image 124 in
As described above, the image which provides the respective probe spots supplied under the optimal exposure conditions can be generated.
As shown in
If it is determined according to the analysis result that some probe spots should not be analyzed since the shape of the probe spots is not circular or impurities exist in the region of the probe spots, a user may click the right button of the mouse on the region of the probe spot to display an adjustment window, and can change the state of the probe spot into an unused probe spot as shown in
FIGS. 17 to 21 illustrate schematic control flowcharts according to the display method of the invention.
First, a user selects a folder which stores the measurement information to be analyzed (step 200). Then, the measurement information having the analysis result is displayed on the screen (step 205). If a user desires to change the display content (step 207), they can select the content to be changed. Then, a user completes the analysis (step 208) by making a selection, thereby storing the analysis result.
Next, the order of operation including a process in the software is that a user first selects the folder storing the measurement information having the analysis result (step 200), then the processor 104 reads the measurement information of the particular test sample from the storage unit 100 (step 201). At step 202, the measurement result analysis unit 104-1 calculates the normalization coefficients based on the signal intensity raw data. Then, the data is normalized at step 203. After the foregoing operations are completed, the measurement result analysis unit 104-1 analyzes the measurement result such as calculating the amount of nucleic acid or the amount ratio of nucleic acid based on the signal intensity data at step 204. The display unit 101 displays the measurement information including the analysis result thereon at step 205. If a user inputs a command to store the measurement information including the analysis result of the measurement result at step 206, the processor 104 stores the measurement information including the analysis result in a predetermined folder of the measurement information storage unit 105. If a user inputs a command to change the display method at step 207 after the analysis result is displayed, the display method selection unit 104-2 changes the display method according to a user's command. The display unit 101 displays the measurement information including the analysis result reflecting a change. If a user inputs a command to finish the analysis at step 205, the processor 104 determines whether the analysis result is stored. If the analysis result is stored a user checks whether the display method is changed. If it is determined that the analysis result is not stored or the analysis result is not stored after the displayed method is changed, the processor 104 determines whether to store the measurement information including the analysis result of the measurement result at step 210. If a user desires to store the measurement information and inputs a command, the processor 104 stores the measurement information including the analysis result in the predetermined folder of the measurement information storage unit 105, thereby completing the storage operation.
FIGS. 18 to 21 are detailed examples of the flowchart in
The table can be rearranged according to ascending or descending order by clicking a reference row. The rows can be rearranged by dragging the items. The table may be arranged by clicking the reference row or dragging the items. If a user inputs a command to rearrange the table, the display method selection unit 104-2 rearranges the table (step 304). A user can select whether to use the data or not and whether to use the data for the normalization or not. This setting may be changed by clicking the right button of the mouse on the selected row, displaying the selection box and clicking the corresponding item therefrom. Then, the command to change the setting is input, and the display method selection unit 104-2 changes the setting (step 305).
A user can set whether to use the data by setting the threshold value. A user may sequentially click “set up” and “analysis condition” items from the menu and input the threshold value with respect to the displayed window to set the threshold value. Then, the command to set the threshold value is input, and the measurement result analysis unit 104-1 sets the threshold value (step 303).
The type of the replicate image is changed by sequentially clicking “display” and “replicate image” items from the menu and clicking the desired replicate image (the replicate image of test sample 1, the replicate image of test sample 2 and the combined replicate image of test samples 1 and 2). Then, the command to display the replicate image is input, and the display method selection unit 104-2 displays the replicate image. The display signal intensity of the replicate image is changed by clicking “display setting” item. The command to change the display signal intensity is input, and the display method selection unit 101-2 changes the display signal intensity (step 313). The type of the data used to display the analysis result is changed by adjusting the ON/OFF state of the raw data display. The command to display the changed image is input and the display method selection unit 104-2 displays the changed image (step 314).
The items “display” and “ratio diagram” are sequentially clicked from the menu to select the test sample as the denominator, to set the sequence of the bar graph and scales of the vertical and horizontal axes (e.g. setting the ON/OFF state to display a logarithmic axis and inputting values of the display range from the window of the display setting). Then, the command to change the foregoing elements is input, and the display method selection unit 104-2 changes the foregoing elements.
A hybrid image which combines the images of the respective probe spots under the optimal signal detecting condition with respect to the two test samples can be displayed by sequentially clicking the items “display” and “image display” from the menu. Then, the command to display the image is input, and the display method selection unit 104-2 displays the hybrid image.
The bibliographic facts, the measurement conditions and so on may be displayed by sequentially clicking the items “display” and “experiment information display” from the menu. Then, the command to display the experiment information is input, and the display method selection unit 104-2 displays the experiment information.
According to the present exemplary embodiments the method of displaying the analysis result of nucleic acid as the biologically relevant material by using the microarray is provided. If the test results of multiple items are displayed, the detection method is not limited to the microarray. The display method can be applicable to display the analysis result of other biologically relevant materials such as hormones, a tumor marker, an enzyme, an antibody, an antigen, an abzyme, other proteins, a nucleic acid, CDNA, DNA, mRNA, etc. Particularly, the method is effective for preparation, analysis, and display of a microarray.
According to the display method of the aspect (1) in which a replicate image which illustrates a layout of a probe spot in the microarray is displayed as the measurement information, the amount of the biologically relevant material or the amount ratio of the biologically relevant material among the plurality of test samples may be represented with color gradation or the types of color tones. The position of the probe spot on the microarray or the normalized, biologically relevant material, e.g. the type of a normalized gene, may be displayed to be easily recognized. Further, information on the bibliographic facts may be displayed. Thus, necessary information may be displayed rapidly, sufficiently, moderately and easily. The normalized gene may include a gene (internal control gene) which is included in the test sample, and a gene (external control gene) which is not included in the test sample.
According to the display method of the aspect (2) in which the amount of the biologically relevant material of the test sample which is measured by using the microarray is normalized to compare the amount of the biologically relevant material in the each test sample, a bias of data due to differences in the fabricated test samples may be normalized. The display method of the invention may display the normalized data to be analyzed easily and efficiently. The optimal normalization method is selected depending on the type of data, thereby analyzing the data more precisely. According to the present display method, the normalization method may be adjusted from any analysis result displayed on a screen. The optimal normalization method is easily selected to analyze the microarray data by displaying the employed normalization method and normalization coefficients.
From a folder storing the measurement information including the analysis result, the serial number of the microarray, the name of experimenter, the type of the optical filter, the measurement condition such as the measurement temperature, or the amount of the biologically relevant material and the ratio of the biologically relevant materials among the plurality of the test samples may be obtained according to the aspect. Thus, a user may recognize the data analysis result under the predetermined experiment condition simultaneously since the foregoing measurement information is displayed.
According to the display method of the aspect (3) in which a bar graph illustrates the amount ratio of the biologically relevant material of two test samples to be compared with each other, the amount ratio of the biologically relevant material is represented with the bar graph. The different amount ratios of the biologically relevant material may be identified by varying the gradation of the bar graph according to the ratio. Then, a user may recognize whether the amount of the biologically relevant material is changed, based on the gradation of the bar graph. Thus, the comparison result is displayed to be viewed easily at a glance.
According to the display method of the aspect (4) in which the relationship between the position of the probe spot and the amount ratio or signal intensity of the biologically relevant material among the two test samples to be compared, with respect to the plurality of probe spots on the microarray, is displayed with a polygonal line graph or a plot changes in the amount ratio or the signal intensity of the biologically relevant material are displayed easily with the polygonal line graph or the plot.
Some probe spots on the microarray are bright while others thereon are dark. Since images which are obtained under the same signal detecting condition are too dark or too bright, the state of the respective probe spots are not recognized precisely. According to the display method of the aspect (5) in which an image which is a combination of images generated under optimal signal detecting conditions for the each probe spot is displayed, however, the image which is a combination of the images generated under the optimal signal detecting condition to the respective probe spots is displayed. Thus, the overall probe spots may be analyzed under optimal conditions.
In consideration of the number of graphs that can be displayed on a monitor and the number of graphs recognizable by a person at a time, the present invention may be applicable to the analysis of 500 items or less (in case of the microarray, the number of analyzed items corresponds to the number of analyzed probe spots. That is, two analyzed items correspond to two equivalent probe spots).
According to the present invention, data or information required for the analysis are displayed through a plurality of display methods, so that a user may view the data or the information and analyze the microarray having a limited number of genes without difficulty. As the data or the information analysis is implemented without difficulty through the present display method, time analyzing the data is saved and the analysis precision may be improved.
Further, the invention is useful in a technical field detecting a biologically relevant material, particularly plural genes or expression of plural genes in plural biological samples.
Number | Date | Country | Kind |
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2005-097893 | Mar 2005 | JP | national |
This application is continuation application of a PCT Application No. PCT/JP2006/306260, filed Mar. 28, 2006, whose priority is claimed on Japanese Patent Application No. 2005-97893, filed Mar. 30, 2005. The description thereof is incorporated herein by reference.
Number | Date | Country | |
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Parent | PCT/JP2006/306260 | Mar 2006 | US |
Child | 11864188 | Sep 2007 | US |