JP2004135663A - Method for analyzing gene expression - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for analyzing gene expression, capable of analyzing the gene expression at a high rate with good accuracy, by using a three-dimensional micro-array. <P>SOLUTION: This method for analyzing the gene expression comprises analyzing a state of the gene expression by using the micro-array in which two or more minute liquid-receiving parts capable of three-dimensionally receiving the liquid are two-dimensionally arranged. Further, the method includes a process for preparing a labeled nucleic acid by using a RNA as a template, another process for positioning a DNA probe which complementarily combines with a part of the labeled gene on the liquid-receiving part, a third process for contacting a liquid sample which contains the labeled nucleic acid with the DNA probe under a condition that a hybrid is formed between the labeled nucleic acid and the DNA probe by making the liquid sample flow inside and outside the micro-array, and the other process for detecting signal strength which changes based on difference of combining strength of the hybrid under another condition that formation of a nonspecific hybrid is dissociated. <P>COPYRIGHT: (C)2004,JPO

Description

(4) The present invention relates to a method for analyzing gene expression, and more specifically, to a method for performing gene expression analysis using a three-dimensional microarray.

In recent years, a molecular biological method called a microarray (DNA chip) has been developed to simultaneously analyze the expression status of a plurality of genes. Microarrays include those in which a large number of cDNA probes are immobilized on the surface of a glass substrate, and those in which a large number of oligo probes are synthesized in a minute area on silicon by applying a semiconductor manufacturing process. From the data obtained using microarrays, it is possible to classify (cluster) many genes into multiple functional groups, and accumulate information on gene fluctuations associated with all life phenomena such as development, differentiation, and aging. Is coming. The gene expression information thus obtained has been used as a database that can be easily accessed through the Internet.

However, gene expression analysis using a microarray can analyze a large number of target genes at once, but requires a long assay time, a large number of overall experimental operation steps, and requires complicated operations. Therefore, there is a disadvantage that an analysis result with good reproducibility cannot be obtained.
As a solution to such a drawback, a method using a microfabricated flow-through porous substrate as a carrier for a microarray (for example, see Patent Document 1), a method for forcibly performing a hybridization reaction by electric force (for example, Patent Document 2) has been developed.

However, in the method using a microfabricated flow-through porous substrate, a reaction part for performing liquid movement and temperature control and a measurement part for performing fluorescence detection are separate, and a complicated apparatus configuration is required. Was very poor.
In the method of forcibly performing the hybridization reaction by electric force, the hybridization reaction can be performed in a short time, but a complicated mechanism for strictly controlling the voltage in the microarray must be provided. was there. For this reason, the microarray and the device have been increased in size, leading to an increase in manufacturing cost, and it has been difficult to put the device into practical use.

Furthermore, on the other hand, regarding the amount of immobilization of the probe on the conventional two-dimensional substrate surface, each probe spot has a non-negligible variation, which makes it difficult to use a microarray particularly for quantitative analysis. There is a problem that there is. In view of such circumstances, by using a metal oxide having a porous structure as a reaction carrier and driving the sample solution from one surface to the other surface, the diffusion speed of the sample solution is increased and the reaction is performed. A method of increasing the speed and performing high-speed and accurate hybridization is disclosed (for example, see Patent Document 3).
Japanese Patent Publication No. 9-504864 JP-T-2001-501301 JP 2000-515251 A

However, in the case where the probe is immobilized on the conventional two-dimensional substrate surface, and in the case where the probe is immobilized on the reaction carrier having the three-dimensional structure having the porous structure as described above, the labeled nucleic acid is in the first place. The environment of the hybridization reaction between the probe and the probe is greatly different. In addition, since the detection is performed while the solution is flowing in the reaction carrier having the three-dimensional structure, setting the reaction conditions is considered to be more complicated. In order to accurately and quantitatively detect gene expression analysis, it is necessary to examine various reaction conditions in detail, but in combination with the difference in the structure of the reaction carrier and the flow of the solution, there is no It is unknown. The prior art does not teach or suggest any specific guidelines for setting the reaction conditions.

Also, a DNA microarray using a reaction carrier having a three-dimensional porous structure is disclosed, but only a few examples of conditions for a hybridization reaction using the DNA microarray are given. Examples of the nucleic acid sample include only an RNA gene (HIV-1) having a length of 145 bases, and hybridization is performed only in a phosphate buffer at room temperature, and has a three-dimensional structure. Only when a single base mutation of a gene is analyzed using a reaction carrier having a porous structure, it cannot be said that the hybridization reaction conditions for gene expression analysis are sufficiently disclosed.

As described above, none of the above methods has been able to meet the demand for a simple and easy analysis of the gene expression state.
Accordingly, the present invention provides a method for performing high-speed and high-accuracy gene expression analysis by using a microarray in which a plurality of microscopic liquid storage units capable of three-dimensionally storing liquids are two-dimensionally arranged. With the goal. More preferably, it is also an object to provide a gene expression analysis method for increasing the signal intensity indicating hybridization between a nucleic acid and a probe.

The gene expression analysis method of the present invention is a method for analyzing the expression state of a gene using a microarray comprising a plurality of two-dimensionally arranged microscopic liquid storage units capable of three-dimensionally storing a liquid, A step of preparing a labeled nucleic acid using RNA as a template, a step of arranging a DNA probe that complementarily binds to a part of the target gene in a liquid container, and a step of preparing a liquid sample containing the labeled nucleic acid inside and outside the microarray. Flowing and contacting the liquid sample with the DNA probe under conditions to form a hybrid between the labeled nucleic acid and the DNA probe, and a signal intensity that changes based on the difference in the binding strength of the hybrid. Detecting the formation of non-specific hybrid under dissociation conditions.
Further, the base length of the DNA probe is preferably 30 to 70 mer.
In addition, it is preferable to heat the liquid sample at 60 to 100 ° C. for 2 to 10 minutes before bringing the liquid sample into contact with the DNA probe.
In addition, it is preferable that the temperature condition for detecting the hybridization reaction between the labeled nucleic acid and the DNA probe and detecting the signal intensity is 35 to 70 ° C.
Further, the hybridization reaction between the labeled nucleic acid and the DNA probe is preferably performed in an SSPE solution having a salt concentration of 0.1 to 6 times.
Further, it is preferable to adjust the pH of the reaction solution at the time of the hybridization reaction between the labeled nucleic acid and the DNA probe to 6 to 8.
In the step of contacting the liquid sample with the DNA probe, the liquid sample is preferably intermittently flowed 30 to 200 times.
Further, after the step of bringing the liquid sample into contact with the DNA probe, it is preferable to remove a non-specific hybrid and an unreacted liquid sample by flowing an SSPE solution having a salt concentration of 0.1 to 6 times inside and outside the microarray.
Further, the pH of the washing solution for removing the non-specific hybrid and the unreacted solution is preferably 6 to 8.

According to the gene expression analysis method of the present invention, the expression state of a gene can be analyzed at high speed and with high accuracy using a three-dimensional microarray. Furthermore, from the viewpoints of workability and cost, the gene expression analysis method of the present invention is highly practical, and is very useful in gene diagnosis, which is expected to be practical in the future, such as disease prediction and diagnosis, virus typing, and the like. You can say that.

The microarray (hereinafter, referred to as a three-dimensional microarray) used in the gene expression analysis method of the present invention may be a microarray in which a plurality of microscopic liquid storage units capable of three-dimensionally storing liquids are two-dimensionally arranged. For example, although there is no particular limitation, an example of the material of the liquid storage unit is, for example, aluminum oxide. Here, the liquid container is a minimum unit for fixing the DNA probe, and is also called a probe spot.

A three-dimensional microarray can immobilize a larger amount of DNA probes than a microarray in which DNA probes are immobilized on a slide glass (hereinafter, a two-dimensional microarray). Further, in conventional two-dimensional microarrays, it is difficult to quantitatively immobilize DNA probes, so that the immobilized amount of each DNA probe often varies. On the other hand, the three-dimensional microarray is suitable for quantitative experiments such as analysis of the expression level, since the DNA probe can be fixed quantitatively.

遺 伝 子 The gene expression analysis method of the present invention is performed using the above three-dimensional microarray, and includes the following steps. That is, a step (1) of preparing a labeled nucleic acid using RNA as a template, a step (2) of arranging a DNA probe complementary to a part of a target gene in a liquid container, (3) contacting the liquid sample with the DNA probe under conditions that allow the liquid sample containing the DNA to flow in and out of the microarray to form a hybrid between the labeled nucleic acid and the DNA probe; (4) detecting a signal intensity that changes based on the difference under the conditions that dissociate the nonspecific hybrid formation.

In this specification, “nucleic acid” refers to DNA and RNA.
“Hybrid” refers to a double strand formed between any of the above nucleic acids.
The “signal” is a signal that can be appropriately detected and measured by an appropriate means, and includes fluorescence, radioactivity, chemiluminescence and the like.

In the step (1) of preparing a labeled nucleic acid, RNA is extracted from a tissue, a cell, or the like, and cDNA and cRNA are synthesized using the RNA as a template. The RNA used as a template may be any of mRNA and total RNA.
The method for labeling a nucleic acid is not particularly limited, and a known method such as performing a reverse transcription reaction using a labeled oligo dT primer or a labeled dNTP mix may be employed.
In the present invention, the labeled nucleic acid thus synthesized is dissolved in, for example, sterilized distilled water and used as a liquid sample. As will be described later, a salt or the like can be appropriately added for the purpose of facilitating hybridization with a DNA probe.

In the present invention, in the step (2) of arranging a DNA probe in the liquid container, first, a DNA probe is prepared. The DNA probe is one which binds complementarily to a part of the target gene, and preferably has a sequence complementary to a base sequence specifically present in the target gene. The base length is preferably from 30 to 70 mer, more preferably from 40 to 60 mer. When the base length is less than 30 mer, nonspecific hybridization tends to increase, and when it exceeds 70 mer, it is not preferable from the viewpoint of time and cost required for DNA probe synthesis.
As described above, it is relatively easy to quantitatively dispose and immobilize such a DNA probe in each liquid storage part, and therefore, the amount of DNA probe in each liquid storage part has little variation and excellent reproducibility. Measurements can be performed.

Next, the step (3) of bringing the liquid sample into contact with the DNA probe is performed under conditions that allow the liquid sample to flow inside and outside the microarray to form a hybrid between the labeled nucleic acid and the DNA probe.
As described above, by flowing the liquid sample into and out of the three-dimensional microarray, the liquid sample containing the labeled nucleic acid can be easily flowed into the space of each liquid container, so that the nucleic acid molecules and DNA The probe molecules can be contacted, and hybridization can be performed in a short time.

Hereinafter, preferable conditions for hybrid formation will be described.
The hybridization reaction between the labeled nucleic acid and the DNA probe is preferably performed in an SSPE solution having a salt concentration of 0.1 to 6 times. That is, the hybridization reaction is performed by adding the SSPE solution to the liquid sample so that the salt concentration falls within the above range. Regarding the salt concentration of the SSPE solution, a higher salt concentration contributes to an increase in signal intensity due to easier hybridization, but when the salt concentration exceeds 6 times, the rate of increase tends to decrease. On the other hand, the lower the salt concentration is, the higher the specificity of hybridization is. However, when the salt concentration is less than 0.1 times, it is necessary to lengthen the reaction time.

Further, as shown in Examples described later, it is preferable to adjust the pH of the reaction solution at the time of hybridization of the above nucleic acid and DNA probe to 6 to 8 in order to optimize the signal intensity. If the pH of the reaction solution deviates from this range, the signal intensity may decrease, depending on the type of the substrate, the substrate may be denatured, or it may be gradually dissolved to make it difficult to obtain an accurate signal intensity. This is because a problem may occur.
To satisfy the above preferred salt concentration and pH range, for example, as a composition of an SSPE solution (20 times), 1 M hydrochloric acid is added to 3.0 M sodium chloride, 0.2 M sodium phosphate, and 0.02 M EDTA, The solution adjusted to pH 6.6 to 7.0 can be used for preparing a sample solution.

に お い て In the step of bringing the liquid sample into contact with the DNA probe, it is preferable that the liquid sample be intermittently flowed 30 to 200 times. If the number is less than 30, the frequency of contact between the labeled nucleic acid molecule and the DNA probe molecule is insufficient, so that the hybridization reaction is often not performed sufficiently. On the other hand, if it exceeds 200 times, the efficiency of hybrid formation tends to decrease, although the reaction time becomes longer.

Before the liquid sample is brought into contact with the DNA probe, the liquid sample is preferably heated at 60 to 100 ° C. for 2 to 10 minutes, more preferably at 70 to 95 ° C. for 2 to 5 minutes. Further, it is more preferable to perform rapid cooling after heating the liquid sample. As described above, by treating the liquid sample, the entanglement (intramolecular bond and / or intermolecular bond) of the labeled nucleic acid molecule contained therein is released, and nonspecific hybridization is suppressed.
Further, as a preferable condition for forming a hybrid, it is preferable to set a predetermined temperature range. The temperature condition will be described in detail later.

Next, the step (4) of detecting the signal intensity that changes based on the difference in the binding strength of the hybrid is performed under conditions that dissociate the nonspecific hybrid. Specifically, after the step of bringing the liquid sample and the DNA probe into contact with each other, washing is performed by flowing an SSPE solution having a salt concentration of 0.1 to 6 times inside and outside the microarray. At this time, if the salt concentration of the SSPE solution is less than 0.1 times, specific hybridization may be dissociated. If the salt concentration exceeds 6 times, unreacted labeled nucleic acid contained in the liquid sample may be dissociated. And the possibility of forming a non-specific hybrid with the DNA probe.
As described above, after the hybridization reaction is completed, the nonspecific hybrid and unreacted liquid sample are removed by flowing the SSPE solution having a salt concentration of 0.1 to 6 times inside and outside the microarray, and more accurate measurement is performed. can do.
Further, when the pH of the reaction solution is adjusted to 6 to 8 during the hybridization of the nucleic acid and the DNA probe described above, the pH of the SSPE solution used as a washing solution after the completion of the hybrid reaction is also adjusted to 6 to 8, This is preferable because the signal intensity can be increased.

The temperature conditions for detecting the hybridization reaction and the signal intensity are preferably 35 to 70 ° C, more preferably 45 to 55 ° C. From the viewpoint of the hybridization reaction, when the reaction temperature is lower than 35 ° C., nonspecific hybridization tends to increase. Further, setting the reaction temperature higher is preferable because the specificity of the hybridization reaction is increased, but if the reaction temperature is too high, the time required for hybridization is increased, or even specific hybridization is inhibited. there is a possibility.
In addition, from the viewpoint of detection of signal intensity, when the temperature is lower than 35 ° C., it is difficult to dissociate nonspecific hybridization. On the other hand, when the temperature exceeds 70 ° C., not only non-specific hybridization but also specific hybridization may be dissociated, and the overall hybridization strength tends to decrease. Then, since the overall signal intensity becomes weak, it becomes difficult to detect the signal. Here, the signal is fluorescence, radioactivity, chemiluminescence, or the like added to a nucleic acid molecule hybridized to a DNA probe molecule, and among these, fluorescence is preferred from the viewpoint of cost and ease of handling.
As described above, as a result of considering these circumstances, more accurate measurement can be performed in a short time by setting the temperature condition as described above.

遺 伝 子 The gene expression analysis method of the present invention can be performed using, for example, an apparatus for measuring fluorescence intensity as shown in FIG. The apparatus 1 includes a microscope 3 for observing events in a microarray 2 for reacting a sample, a stage 4 provided on the microscope 3 for supporting the microarray 2, and a microarray 2 observed by the microscope 3. A CCD (charge coupled device) camera 5 for capturing a fluorescent image at the XY stage controller 7 for controlling a motor driver 6 connected to the stage 4 to change the field of view observed by the microscope 3; A pump driver 8 for transporting a fluid through a joint connected to the microarray 2, a temperature controller 9 for adjusting the temperature of the microarray 2 via a heater disposed in contact with the microarray 2, and , Included in the genetic test device 1 Computer 10 which controls are collectively connected to all devices and their input device 15, and a monitor 16.

Although not shown in the figure, the joint portion is connected to the microarray 2 in such a manner that the liquid contained in the microarray 2 does not leak outside. Further, the other end of the joint portion is connected to a pump driver 8 via a tube, and the pump driver 8 is further connected to a reagent holding portion containing a desired reagent or the like therein. The pump driver 8 sends and receives a fluid to and from the microarray 2 according to an instruction from the computer 10 according to an analysis program.
Similarly, although not shown, the heater section is disposed between the microarray 2 and the stage 4. The heater section is connected to a temperature controller 9 and the temperature is controlled by a temperature control 9 according to an instruction from a computer 10.

The computer 10 includes components included in a general computer.
The information captured by the CCD camera 5 is sent to the image processing unit, processed by the image processing unit according to the instruction of the CPU according to the analysis program stored in the memory, and quantified as fluorescence intensity.

The microscope that can be used in the present invention is a commonly used fluorescence microscope, specifically, an Olympus fluorescence microscope AX-70 or BX-52TRF.
The CCD camera that can be used in the present invention is a commonly used CCD camera, and a general digital camera such as a MegaPlus CCD camera manufactured by Kodak, a Cool Snap mono camera manufactured by Roper, or the like can be used.

Here, the temperature control unit for controlling the temperature of the microarray 2 includes a heating element (for example, an electric heater, an electromagnetic heater, a water bath, an air bath, and a Peltier element) for transferring heat by contacting the microarray 2. Etc.), a sensor for sensing the temperature therein, and a temperature controller for controlling the heating in the heating element according to the control of the computer and the information from said sensor.

The stage 4 has its field of view changed by the XY stage controller 7, by the CCD camera 5 itself, or by using various scanning mechanisms for changing the optical path connecting the CCD camera 5 and the microarray 2.

According to such an apparatus 1, since the hybridization reaction, the detection of the signal intensity, and the analysis thereof can be continuously performed, it can be said that the workability is extremely excellent. In addition, a series of operations including a hybridization reaction, detection of signal intensity, and analysis thereof can be performed in about 60 to 120 minutes.

As described above, according to the gene expression analysis method of the present invention, a highly reliable measurement can be performed in a short time. Therefore, it can be said that the present invention is extremely useful not only in biochemical academic applications but also in gene diagnosis, which is expected to be practical in the future, such as disease prediction and diagnosis, virus typing, and the like.

<Preparation of liquid sample>
25 μg each of total RNA derived from a normal human fibroblast cell line TIG-1 (hereinafter abbreviated as TIG-1) and total RNA derived from a human colon cancer cell line COLO320DM (hereinafter abbreviated as COLO320DM) were prepared and used as a template. As a first step, FITC-labeled single-stranded cDNA was prepared by reverse transcription using oligo dT primer. The FITC-labeled cDNA was dissolved in 35 μl of sterile distilled water, denatured by heating at 95 ° C. for 5 minutes, and then rapidly cooled in ice water. To this cDNA solution, 15 μl of a 20 × SSPE solution was added (final salt concentration 6 × SSPE solution) to obtain a liquid sample.

<Design and immobilization of DNA probe>
One gene not present in the human genome (negative control; NC), one gene whose expression is expected to specifically increase in COLO320DM (c-myc), one typical housekeeping gene (β-actin), One gene (GAPDH: internal control; IC) predicted to have no change in the expression level between both cells was selected, and a 60-mer DNA probe was designed based on a nucleotide sequence specific to each cDNA sequence. did. Then, a three-dimensional microarray in which these four types of DNA probes were immobilized on two spots each was prepared.

Each liquid sample was liquid-driven 150 times by the three-dimensional microarray to form a hybrid. The reaction temperature at this time was set to 50 ° C. After the reaction was completed, 50 μl of the 6 × SSPE solution was renewed three times, and the liquid was driven once each to perform washing. After the washing was completed, a fluorescent image was obtained using a WIBA filter cassette (Olympus Optical Industry Co., Ltd.) using a CCD camera mounted on a fluorescent microscope. FIG. 2 (B) shows the results of hybridization using a liquid sample derived from TIG-1, and FIG. 2 (C) shows the results of hybridization using a liquid sample derived from COLO320DM. FIG. 2A shows the arrangement position of each DNA probe.

結果 From the results of FIG. 2 (B) and FIG. 2 (C), no fluorescence was observed in NC (negative control), indicating that the hybridization reaction proceeded specifically. When observing the spots of c-myc and β-actin, it was confirmed that the fluorescence intensity of COLO320DM was certainly higher than that of TIG-1.

Also, the average value of spot luminance was calculated for each gene. This is shown in FIG. When the brightness of GAPDH (IC) was corrected to 1: 1 and the expression level ratio of each gene was compared, COLO320DM showed a 5.5-fold increase in c-myc and a 1.4-fold increase in β-actin. Was done.

Furthermore, in order to verify the results of gene expression analysis on the three-dimensional microarray from different angles, kinetic RT-PCR was performed using total RNA derived from TIG-1 and cDNA synthesized using total RNA derived from COLO320DM as a template. Carried out. The conditions at this time are as follows: a cycle consisting of heat denaturation at 94 ° C. for 5 minutes, annealing at 94 ° C. for 30 seconds, annealing at 63 ° C. for 30 seconds, and extension reaction at 72 ° C. for 30 seconds. It was set to be.
For each gene, PCR was performed so as to obtain a PCR product of about 200 bp including the DNA probe immobilized on the microarray, and agarose electrophoresis was performed on the PCR products at the 15, 18, 21, and 25th cycles. FIG. 4A schematically shows an agarose electrophoresis image of a PCR product obtained using total RNA derived from TIG-1 as a template, and agarose electrophoresis image of a PCR product obtained using total RNA derived from COLO320DM as a template. FIG. 4B schematically shows the electrophoresis image.

As shown in Table 1, the gene expression amount estimated from the number of appearance cycles of the band was 10-fold for c-myc and doubled for β-actin, and the pattern of these gene expression levels was determined by the three-dimensional microarray. And a good correlation was obtained.

From the above results, it was found that the gene expression analysis method of the present invention can measure a highly specific hybridization reaction with high sensitivity.
Further, in FIGS. 2 (B) and 2 (C), DNA probes were arranged at two spots for each gene, but there was almost no difference in the fluorescence intensity of the two spots, so that the reproducibility was excellent. It has been found.

<Preparation of liquid sample>
Three sets of 25 μg each of total RNAs derived from a normal human fibroblast cell line TIG-1 (hereinafter, abbreviated as TIG-1) were prepared, and FITC-labeled single-stranded cDNA was prepared by reverse transcription using oligo dT primer as a template. Produced. This FITC-labeled cDNA was dissolved in 37.5 μl of sterilized distilled water, denatured by heating at 95 ° C. for 5 minutes, and then rapidly cooled in ice water. To this FITC-labeled cDNA solution, 12.5 μl of a 20 × SSPE solution whose pH was adjusted to (1) 6.6, (2) 7.0, (3) 7.4 (unadjusted) was added, and a liquid sample was added. (Final salt concentration 5 × SSPE solution). The pH of each liquid sample was about (1) 7.0, (2) 7.4, and (3) 7.8 (unadjusted).

<Design and immobilization of DNA probe>
Among the cDNAs derived from TIG-1, a three-dimensional microarray in which a 60-mer DNA probe was immobilized one spot at a time on a substrate of an aluminum anodic oxide film based on a base sequence specific to the β-actin and GAPDH genes was used. .

Each liquid sample was liquid-driven 150 times by the three-dimensional microarray to form a hybrid. The reaction temperature at this time was set to 50 ° C. After completion of the reaction, 5 × SSPE solutions having the same pH (1) 6.6, (2) 7.0, (3) 7.4 (unadjusted) as the above 20 × SSPE solution were prepared, and Washing was performed by driving the liquid once each while updating the 5 × SSPE solution three times with 50 μl each. After the completion of the washing, the volume of the 5 × SSPE solution was adjusted to 30 μl, and a fluorescent image was obtained using a CCD camera mounted on a fluorescent microscope using a WIBA filter cassette (manufactured by Olympus Optical Co., Ltd.). Table 2 shows the results of measuring the spot luminance based on this fluorescent image.

Liquid samples were prepared using 20 × SSPE solutions at pH (4) 5.2 and (5) 8.1. The pH of the liquid sample was (4) 5.6 and (5) 8.4. Hybridization was performed using these liquid samples in the same manner as above except for the conditions described above.If liquid driving was repeated under either condition, the substrate could be denatured and accurate measurement was possible. Did not.
As shown in Table 2, when the pH of the sample solution was adjusted within the range of 6 to 8, an accurate signal could be obtained without denaturing the substrate. Further, when the pH of the liquid sample was adjusted to 6.8 to 7.4, an optimum signal intensity was obtained. Therefore, it was found that by performing the hybridization reaction between the nucleic acid and the DNA probe and washing within this pH range, a more accurate gene expression analysis result can be obtained.

FIG. 2 is a schematic diagram showing an apparatus for performing the gene expression analysis method of the present invention. FIG. 2 (A) shows the arrangement of each DNA probe, and FIG. 2 (B) is a schematic diagram of a hybridization result (fluorescence image) when a TIG-1-derived liquid sample is used, and FIG. () Is a schematic diagram of a hybridization result (fluorescence image) when a liquid sample derived from COLO320DM is used. It is a bar graph which shows the average value of spot luminance. FIG. 4 (A) shows electrophoresis showing the amounts of PCR products at the 15, 18, 21, and 25th cycles when performing kinetic RT-PCR using cDNA synthesized using TIG-1-derived total RNA as a template. FIG. 4 (B) is a schematic diagram of the image, and FIG. 4 (B) shows the amounts of PCR products at the 15, 18, 21, and 25th cycles when kinetic @ RT-PCR was performed using cDNA synthesized with total RNA derived from It is a schematic diagram of the electrophoretic image shown.

Explanation of reference numerals

DESCRIPTION OF SYMBOLS 1 ... Device 2 ... Microarray 3 ... Microscope 4 ... Stage 5 ... CCD camera 9 ... Temperature controller 10 ... Computer

Claims (9)

A method for analyzing the expression state of a gene using a microarray in which a plurality of microscopic liquid storage units capable of storing liquid three-dimensionally is arranged two-dimensionally,
Using RNA as a template to produce a labeled nucleic acid,
Arranging a DNA probe complementary to a part of the target gene in the liquid container,
Flowing a liquid sample containing the labeled nucleic acid inside and outside the microarray, and contacting the liquid sample with the DNA probe under conditions that allow a hybrid to form between the labeled nucleic acid and the DNA probe;
Detecting a signal intensity that changes based on a difference in hybrid binding strength under conditions that dissociate nonspecific hybrids. 請求 The gene expression analysis method according to claim 1, wherein the DNA probe has a base length of 30 to 70 mer. (3) The gene expression analysis method according to (1) or (2), wherein the liquid sample is heated at 60 to 100 ° C. for 2 to 10 minutes before bringing the liquid sample into contact with the DNA probe. The gene expression according to any one of claims 1 to 3, wherein the temperature condition for detecting the hybridization reaction between the labeled nucleic acid and the DNA probe and the signal intensity is 35 to 70 ° C. analysis method. The method according to any one of claims 1 to 4, wherein the hybridization reaction between the labeled nucleic acid and the DNA probe is performed in an SSPE solution having a salt concentration of 0.1 to 6 times. . (6) The method according to any one of (1) to (5), wherein the pH of the reaction solution during the hybridization reaction between the labeled nucleic acid and the DNA probe is adjusted to 6 to 8. The method according to any one of claims 1 to 6, wherein in the step of contacting the liquid sample with the DNA probe, the liquid sample is intermittently flowed 30 to 200 times. After the step of contacting the liquid sample with the DNA probe, a non-specific hybrid and an unreacted liquid sample are removed by flowing an SSPE solution having a salt concentration of 0.1 to 6 times inside and outside the microarray. The gene expression analysis method according to claim 1. The gene expression analysis method according to any one of claims 1 to 8, wherein a pH of the washing solution for removing the non-specific hybrid and the unreacted solution is 6 to 8.

Images