JP2004226255A - New microarray - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microarray for performing the manifestation analysis of an abnormal protein and that of genes with a small number of samples as much as possible, and to provide a manifestation analysis method of protein and genes using the microarray. <P>SOLUTION: The manifestation analysis method of protein or genes features the microarray, where a hydrophobic sheet is applied to the contour section (peripheral section) of a section in an array on slide glass in which antibodies or DNAs are arrayed, and the use of the microarray. Additionally, the method features the microarray, where the hydrophobic sheet is applied by allowing a gap that may become a sample injection opening to remain at the contour section (peripheral section) of a part in an array on the slide glass in which the antibodies or DNAs are arrayed, especially the microarray, where the upper surface of a part in which at least the antibodies or DNAs on the slide glass are arrayed, is covered with cover glass, and the use of the microarray. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel microarray in which the required sample amount is reduced, and which can be used for, for example, expression analysis of abnormal proteins such as cancer, gene expression analysis, and the like.
[0002]
[Prior art]
In the case of an existing protein antibody array (for example, Clontech Antibody Microarrays), at least 1 mL of a protein having a protein mass of 1.1 mg / mL is required for performing one antibody experiment. Requires a cell amount and a tissue amount (untreated tissue) of up to 150 mg. For example, in a cultured cell, depending on the type of the cell, about 4 to 5 cells are required in a 100 mm petri dish, and the amount of tissue, for example, in the case of a cancer cell or the like, cancer is reliably determined from each organ of a human. It is necessary to collect 150 mg of such a site, which corresponds to almost the entire amount of highly advanced cancer. Depending on the method of harvesting and collection, too many normal tissues may be included, requiring a large amount of specimen.
In addition, although the amount of normal tissue as a control has never exceeded the collection amount as small as possible, the amount of cancer that can be obtained from a single animal in a mouse model or the like is limited, and the number of individuals is large. When mixed, there is a problem that the effect of the solid is not understood and the average value is obtained.
One of the reasons that the existing antibody array requires a large amount of sample is that when the antibody spotted on the slide glass reacts with the protein as the specimen, a protein-containing solution is placed in an incubation tray, and the For dissolving the protein in about 5 mL of buffer to immerse the slide glass in the reaction.
[0003]
[Problems to be solved by the invention]
The present invention has been made in view of the current situation as described above, and provides a microarray capable of performing abnormal protein expression analysis and gene expression analysis with a sample amount as small as possible, and a protein and gene expression analysis method using the same. The purpose is to provide.
[0004]
[Means for Solving the Problems]
The present invention relates to a microarray in which a hydrophobic sheet is attached to an outer peripheral portion (peripheral portion) of an arrayed portion on a slide glass on which antibodies or DNA are arrayed.
[0005]
The present invention also provides a method in which a hydrophobic sheet is attached to an outer peripheral portion (peripheral portion) of an arrayed portion on a slide glass on which an antibody or DNA is arrayed, leaving a gap that can be used as a sample injection port. The present invention relates to an attached microarray.
[0006]
Furthermore, the present invention is characterized in that a microarray is used in which a hydrophobic sheet is attached to an outer peripheral portion (peripheral portion) of an arrayed portion on a slide glass on which antibodies or DNAs are arrayed. The present invention relates to a method for analyzing the expression of a protein or gene to be expressed.
[0007]
Furthermore, the present invention provides a hydrophobic sheet by leaving a gap that can serve as a sample inlet at an outer peripheral portion (peripheral portion) of an arrayed portion on a slide glass on which an antibody or DNA is arrayed. The present invention relates to a method for analyzing the expression of a protein or a gene, which comprises using an attached microarray.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
The microarray of the present invention is, for example, as shown in (A) (top view) and (B) (side view) of FIG. 1, on a slide glass on which an antibody or DNA (single-stranded DNA) is arrayed. One or more hydrophobic sheets are attached to the outer periphery (peripheral portion) of the arrayed portion, and the feature is that the arrayed portion of the antibody or DNA is pooled.
1 (A) and 1 (B), 1 is a slide glass, 2 is a portion where an antibody or DNA is arrayed, 3 is a hydrophobic sheet, and 4 is a spot of an antibody or DNA.
[0009]
Further, another type of microarray of the present invention is, for example, as shown in FIG. 2A, an arrayed portion on a slide glass on which an antibody or DNA (single-stranded DNA) is arrayed. Is characterized in that one or a plurality of hydrophobic sheets are adhered to the outer part (peripheral part), leaving a gap that can be a sample injection port.
This type of microarray usually covers at least the upper surface of the slide glass on which the antibody or DNA is arrayed with a cover glass or the like (when at least the antibody or DNA is arrayed on the slide glass). FIG. 2 is a side view (cross-sectional view) of the case where at least the upper surface of the arrayed portion is covered with a cover glass or the like. Shown in B).
2 (A) and 2 (B), 1 is a slide glass, 2 is a portion where an antibody or DNA is arrayed, 3 is a hydrophobic sheet, 4 is a spot of antibody or DNA, 5 is a sample injection port ( (A gap portion), 6 is a cover glass or the like, and 7 is a minute space that can accommodate a sample.
[0010]
Examples of the hydrophobic sheet used in the present invention include a plastic sheet, a laminated paper, and a silicone sheet.
As a material of the plastic sheet, for example, polyvinyl chloride, polyethylene, polypropylene and the like can be cited as typical examples. However, any material is usually used as long as it is used for sheets, seals, tapes, patches and the like. It may be something.
Examples of the laminate material of the laminated paper include, but are not limited to, polyethylene, polypropylene, aluminum foil, and the like, and are not limited thereto, and may be a laminate material such as moisture-proof, oil-resistant, heat-sealing, or chemical-resistant. Any material having the necessary characteristics may be used.
Further, as the silicone sheet, a sheet made of silicone resin or silicone rubber or the like corresponds thereto.
[0011]
As these hydrophobic sheets used in the present invention, it is convenient and preferable to use those in the form of a so-called pressure-sensitive adhesive sheet or pressure-sensitive adhesive tape in which a pressure-sensitive adhesive has been applied to one side (or both sides) in advance. It goes without saying that the adhesive may be applied to a simple sheet or tape having no adhesive applied, and then used.
Also, when a sheet made of ultra-low hardness silicone rubber or the like is used as the silicone sheet, there is no particular need to use an adhesive or the like since the sheet itself has tackiness.
The pressure-sensitive adhesive applied to the pressure-sensitive adhesive sheet or pressure-sensitive adhesive tape used in the present invention, or the pressure-sensitive adhesive to be applied to a simple sheet or tape when used, is generally used for general-purpose pressure-sensitive adhesive sheets and pressure-sensitive adhesive tapes Examples include, but are not limited to, rubber-based elastomers and acrylic resins, and the hydrophobic sheet attached to the slide glass can be relatively easily peeled off from the slide glass, and after peeling. Any pressure-sensitive adhesive may be used as long as substantially no material remains on the slide glass.
[0012]
The hydrophobic sheet used in the present invention is attached to the outer portion (peripheral portion) of the arrayed portion without leaving a gap that can serve as a sample injection port, and the portion where the antibody or DNA is arrayed is attached. When used as a pool, a plurality of sheets are stacked as necessary so that the thickness of the sheet portion is 0.5 to 2 mm, preferably 0.5 to 1.5 mm, more preferably about 1 mm. It is used, but if one sheet has such a thickness, one sheet may be used.
When a hydrophobic sheet is attached to the outer peripheral portion (peripheral portion) of the arrayed portion while leaving a gap that can be used as a sample injection port, and the upper surface thereof is covered with a cover glass or the like, the sheet is used. Since the thickness of the portion is preferably 0.05 to 0.5 mm, and preferably 0.1 to 0.2 mm, a plurality of such layers may be used if necessary so as to have such a thickness. If there is such a thickness, of course, one sheet may be used.
[0013]
The width and length of the hydrophobic sheet used in the present invention are naturally determined by the space of the outer peripheral portion (peripheral portion) of the arrayed portion on the slide glass on which the antibody or DNA is arrayed.
That is, for example, when there is a 25 mm × 25 mm arrayed portion at the top on a 30 mm × 65 mm × 1 mm slide glass, the width of the hydrophobic sheet is 2.5 mm, and the length is the length of the sample. This is a length that can surround the periphery of the arrayed portion of 25 mm × 25 mm with or without a gap that can serve as an inlet.
[0014]
As the hydrophobic sheet used in the present invention, it is also possible to conveniently use a product display sticker or the like attached to a bottle of food or the like.
In other words, these product display stickers were not easily peeled off because they were previously adhered using an adhesive, but recently there are some types that can be peeled off neatly. If there is a seal larger than the part which is arranged, an area corresponding to the part arranged in the array may be cut out and attached to the outer part (peripheral part) of the part arranged in the array.
[0015]
The microarray of the present invention leaves a gap that can serve as a sample injection port in an outer portion (peripheral portion) of an arrayed portion on a slide glass on which an antibody or DNA (single-stranded DNA) is arrayed. Alternatively, it is characterized in that a hydrophobic sheet is attached without leaving it, but has a portion where an antibody or DNA (single-stranded DNA) is arrayed, to which a hydrophobic sheet is attached. As a slide glass, a microarray in which an antibody (single-stranded DNA) or an antibody (single-stranded DNA) is arrayed, which is already (commercialized, etc.), may be used on a slide glass having been subjected to an appropriate surface treatment. An antibody or DNA (single-stranded DNA) may be appropriately spotted according to the purpose and arrayed.
[0016]
According to the present invention, an antibody or a DNA (single-stranded DNA) is arrayed on a slide glass on an outer peripheral portion (peripheral portion) of the arrayed glass slide, leaving a gap that can serve as a sample injection port. As described above, a microarray formed by attaching an adhesive sheet is usually covered with a cover glass or the like on at least the portion of the slide glass on which the antibody or DNA is arrayed (on the slide glass, A cover glass or the like is used in contact with at least the upper surface of the portion where the antibody or DNA is arrayed), and the cover glass or the like is usually a slide glass similar to the slide glass on which the antibody or DNA is arrayed. Are used. The cover glass or the like may naturally cover not only the upper surface of the portion where the antibody or DNA is arrayed but also the entire upper surface of the slide glass.
[0017]
The hydrophobic sheet 1 is formed on the slide glass on which the antibody or DNA (single-stranded DNA) is arrayed without leaving a gap that can serve as a sample injection port in the outer peripheral portion (peripheral portion) of the arrayed portion. The procedure for performing protein or gene expression analysis using a microarray in which a plurality of sheets are pasted and a pool of antibodies or DNAs are pooled is as follows. The sample solution containing the protein or DNA is injected into the portion where the sample is contained, for example, using a micropipette or a syringe.
In this case, the size of the pool is, for example, 25 mm × 25 mm × 1 mm when the area of the portion where the antibody or DNA is arrayed is 25 mm × 25 mm and the thickness of the hydrophobic sheet is 1 mm. = 625 μL.
Therefore, if the amount of protein / sample solution is conventional (〜50 μg / 5 mL) × 4 batches, even if the sample solution is completely filled in the pool, (6.25 μg / 625 μL) × 4 And the protein amount may be one-eighth of the conventional value, and when the sample solution is put into a height of 0.5 mm, which is half the thickness of the sheet portion, the protein amount may be one-sixteenth of the half. . That is, conventionally, 150 mg of tissue or cells was required, but 10 mg is sufficient.
[0018]
Thus, the sample solution is injected into a pool prepared by attaching one or more hydrophobic sheets to the outer portion (peripheral portion) of the portion where the antibody or DNA (single-stranded DNA) is arrayed, and then slide glass. After reacting (incubating) the antibody or DNA arrayed on the above and the sample solution at a predetermined temperature (normally room temperature) for a predetermined time (normally 30 minutes) while occasionally shaking the microarray, a hydrophobic sheet is applied. Remove and wash slides (microarray after incubation) with appropriate wash buffer. After the washed slide glass is dried by centrifugation or the like, scanning is performed according to a conventional method. The subsequent operation is exactly the same as that of the protein or gene expression analysis method using a normal microarray.
[0019]
In addition, a hydrophobic sheet is left on the outer peripheral portion (peripheral portion) of the arrayed portion on the slide glass on which the antibody or DNA (single-stranded DNA) is arrayed, so as to leave a gap that can serve as a sample injection port. The procedure for performing protein or gene expression analysis using the microarray of the present invention in which the top surface of the portion where the antibody or DNA is arrayed is covered with a cover glass is first performed on a slide glass. From the gap (sample injection port) between the hydrophobic sheets attached to the outer part (peripheral part) of the arrayed part, a space surrounded by a slide glass, a hydrophobic sheet, a cover glass, etc. A sample solution containing protein or DNA is injected using a micropipette or a syringe.
In this case, for example, the area of the space where the antibody or DNA is arrayed is 25 mm × 25 mm, and the thickness of the hydrophobic sheet is 0. 1 mm, 25 mm × 25 mm × 0. 1 mm = 62.5 μL.
Therefore, while the amount of protein / sample solution is (試 料 50 μg / 5 mL) × 4 batches in the conventional case, even if the sample solution is completely filled in the space, (0.625 μg / 62.5 μL) ) × 4, and the protein amount may be 1/80 of the conventional amount. That is, 2 mg is sufficient instead of 150 mg of tissue or cells conventionally required.
[0020]
Thus, after injecting the sample solution into the space surrounded by the slide glass, the hydrophobic sheet, and the cover glass, the antibody or DNA arrayed on the slide glass and the sample solution are heated to a predetermined temperature while occasionally shaking the microarray. After the reaction (incubation) for a predetermined time (normally 30 minutes) at (normal room temperature), the cover glass and the hydrophobic sheet are removed, and the slide glass (microarray after incubation) is washed with an appropriate washing buffer. After the washed slide glass is dried by centrifugation or the like, scanning is performed according to a conventional method. The subsequent operation is exactly the same as that of the protein or gene expression analysis method using a normal microarray.
[0021]
In either case, if a sheet that can be easily peeled off as a hydrophobic sheet and does not leave an adhesive or the like is used, it will not interfere with scanning. When a sheet or adhesive made of a material that does not interfere with the measurement is used, it is not necessary to remove the sheet after the incubation.
[0022]
The method for preparing a sample solution to be used in the method for analyzing protein or gene expression using the microarray of the present invention is the method for preparing a sample solution to be used in the method for analyzing protein or gene expression using a normal microarray. The method may be exactly the same.
That is, for example, when performing protein expression analysis, a protein is extracted from cells or tissues (untreated tissues) according to a predetermined operation procedure, the protein concentration is measured, and then the protein is diluted to a predetermined concentration.
Next, the protein in this solution is labeled with a fluorescent dye (for example, Cy3, Cy5, etc.) or the like, and the unbound dye or the like is removed to obtain a sample solution.
When gene expression analysis is performed, for example, total RNA or the like is isolated from cells or tissues (untreated tissues) according to a predetermined operation procedure, and the yield and purity are analyzed. A probe is prepared by labeling with Cy3, Cy5, etc.) or a radioactive substance. This is dissolved in an appropriate buffer to obtain a sample solution.
[0023]
As described above, the protein or gene expression analysis method according to the present invention uses the microarray of the present invention as a microarray, except that the incubation is performed by the method described above, from preparation of a sample solution to scanning of the microarray. The procedure can be carried out in exactly the same procedure as that of a method for analyzing the expression of a protein or gene using a microarray, which is known per se.
[0024]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
[0025]
Reference Example 1
(1) Sample preparation
Jalkat cells cultured in RPMI medium (10% FBS buffer) were 1 × 10 ⁷ Each was dispensed, replaced with a HEPES buffer, pre-cultured at 37 ° C. for 1 hour, and then heat-treated at 37 ° C. or 51 ° C. for 30 minutes (hereinafter, each sample is referred to as sample A and sample B). .
Each sample was washed four times with PBS (manufactured by Sigma) (30 mL was used for one wash), the supernatant was sufficiently removed, and then frozen once in a -80 ° C deep freezer.
[0026]
(2) Protein extraction
Each sample was returned to room temperature, and 20 μL × 150 of Extraction / Labeling Buffer provided in the kit of Ab Microarray 380 (manufactured by Clontech) was added, followed by vortex mixing. After shaking at room temperature for 10 minutes, the suspension was centrifuged at 4 ° C. for 30 minutes, and the supernatant was used as a protein sample. The protein concentration was adjusted to 1.1 mg / mL with a BCA reagent (BCA protein Assay Kit: manufactured by Pierce).
[0027]
(3) Fluorescent dye labeling
The amount of dye for 1 mg of each of Cy5 mono-Reactive Dye Pack and Cy3 mono-Reactive Dye Pack (manufactured by Amersham) is added to Extract / Labeling / Extension / Labeling, which is provided with the kit of Ab Microarray 380 (manufactured by Clontech). Dissolved. After thoroughly mixing with a vortex for 20 seconds, the mixture was centrifuged for 10 seconds, and each 50 μL was dispensed into two 1.5 μL microtubes (hereinafter, these were respectively A-Cy3, B-Cy3, A-Cy5, B- Cy5). Sample A was added to A-Cy3 and A-Cy5, and Sample B was added to B-Cy3 and B-Cy5, respectively. Each microtube was mixed by inversion three times, and then centrifuged for 10 seconds. This was incubated on ice (4 ° C.) for 90 minutes. During this time, each tube was mixed by inversion every 20 minutes. 4 μL of Blocking Buffer provided in the kit of Ab Microarray 380 (Clontech) was added to each tube. This was incubated on ice (4 ° C.) for 30 minutes (inverted every 10 minutes).
[0028]
(4) Purification
Using a Milli-Q grade water, a 10 × Desalting buffer provided in a kit of Ab Microarray 380 (manufactured by Clontech) was diluted to prepare 1 × Desalting buffer 100 mL (pH 7.4). Four PD-10 Desalting Columns (manufactured by Amersham) were prepared, and the column was equilibrated with the 1 × Desalting buffer prepared above. A-Cy3, A-Cy5, B-Cy3, and B-Cy5 samples prepared in (3) were added to the column, 2 mL of 1 × Desalting buffer was added, and 2 mL of 1 × Desalting buffer was added. The permeate was collected in a microtube as purified protein. After purification, the protein concentration was measured with a BCA reagent (BCA protein Assay Kit: Pierce) to obtain 0.1 mg / mL of each purified protein. A-Cy5 and B-Cy3 were mixed, and A-Cy3 and B-Cy5 were each mixed in an amount of 100 μg to prepare antibody reaction protein mixtures A-Cy5 / B-Cy3 and A-Cy3 / B-Cy5.
[0029]
(5) Antibody reaction
An Incubation buffer of 45 mL was prepared by mixing 4.5 mL of Background Reducer provided in a kit of Ab Microarray 380 (manufactured by Clontech) with 40.5 mL of Stock Incubation Buffer. 5 mL of each of the above-prepared incubation buffers was added to two places on a tray provided in a kit of Ab Microarray 380 (manufactured by Clontech). A-Cy5 / B-Cy3 and A-Cy3 / B-Cy5 were added to the antibody reaction protein mixture prepared in step (4) in a total protein amount of 50 μg each. The tray was incubated for 30 minutes at room temperature with shaking.
The Ab Microarray (Clontech) slide glass was washed using a stock incubation buffer and an incubation buffer attached to the Ab Microarray 380 (Clontech) kit. The slide glass of the antibody array after washing was put into an incubation tray one by one, and incubated at room temperature for 30 minutes.
[0030]
(6) Cleaning of slide glass
The Incubation buffer provided in the kit of Ab Microarray 380 (manufactured by Clontech) was placed in the remaining two portions of the tray by 5 mL each, and the slide glass after the reaction was transferred and immersed, and incubated at room temperature for 5 minutes. Using Wash buffers 1 to 7 provided in the kit of Ab Microarray 380 (manufactured by Clontech), washing was performed for 5 minutes each while changing the buffer sequentially. After washing, the slide glass was dried by centrifugation.
[0031]
(7) Scanning and data analysis
Scanning was performed using a microarray scanner of Agilent G2565AA (manufactured by Agilent). Analysis was performed using Feature Extraction (manufactured by Agilent) software and array analysis software Gene Pix Pro, and for the labeled protein mixture A-Cy3 / B-Cy5, A (Cy3) in FIG. 3 and FIG. For B-Cy3 / A-Cy5, A (Cy3) in FIG. 5, B (Cy5) and FIG. 6 for B-Cy3 / A-Cy5 were obtained.
[0032]
Example 1
Using an antibody array slide glass in the kit of Ab Microarray 380 (manufactured by Clontech), the outer periphery (peripheral portion) of the antibody array spot was hollowed out so as to surround the antibody array spot, and the thickness was 1 mm. (A) and (b), a silicone sheet (ultra-low-hardness silicone rubber 2 °: manufactured by Sunplatech Co., Ltd.) was closely adhered to form a pool. The ultra-low hardness silicone rubber was sticky and completely adhered to the slide glass. A protein solution was prepared by dissolving the protein mixture for antibody reaction A-Cy5 / B-Cy3 prepared in steps (1) to (4) of Reference Example 1 in 200 μL of Incubation buffer for 2 μg as a total protein amount, and stored in the pool. The mixture was injected using a micropipette ((C) in FIG. 1), and reacted at room temperature for 30 minutes with occasional shaking. On the way, light pipetting was performed to increase the reaction efficiency. After the reaction, scanning was performed using a microarray scanner of Agilent G2565AA (manufactured by Agilent) in the same manner as in Reference Example 1.
The results are shown in FIG. 7 (A) (Cy3) and (B) (Cy5).
As is clear from FIGS. 7A and 7B, sufficient fluorescence intensity was obtained for both Cy3 and Cy5, and the antibody reaction was extremely good.
[0033]
Example 2
Five 28 μm-thick sheets hollowed out so as to surround the antibody array spot, leaving a gap that can be used as a sample injection port, at the outer part (peripheral edge) of the antibody array spot on the slide glass of the antibody array. Thus, a micro pool having a thickness of 140 μm was prepared (FIG. 2A). Next, a cover glass (30 × 30 mm) was closely adhered and fixed thereon to form a minute space (FIG. 2B). A solution prepared by dissolving 0.8 μg as a total protein amount of the protein mixture for antibody reaction A-Cy5 / B-Cy3 prepared in steps (1) to (4) of Reference Example 1 in 80 μL of the incubation buffer was prepared. A micropipette was used to inject into the above-mentioned micro space from the inlet (FIG. 2 (C)), and reacted at room temperature for 30 minutes. Scanning was performed using a microarray scanner of Agilent G2565AA (manufactured by Agilent) as in Reference Example 1.
The results are shown in FIG. 8 (A) (Cy3) and (B) (Cy5).
As is clear from (A) and (B) of FIG. 8, sufficient fluorescence intensity was obtained for both Cy3 and Cy5, and the antibody reaction was extremely good.
[0034]
Since the conventional method uses a tray solution with poor reaction efficiency, 5 ml of the reaction solution is required, and the mixture of the protein samples A and B varies depending on the origin of the sample as the protein weight after labeling purification. Requires 10 μg to 50 μg, whereas the method of Example 1 of the present invention can analyze with a protein amount of 2 μg, and the method of Example 2 can analyze with a protein amount of 0.8 μg. Achieved.
[0035]
【The invention's effect】
The present invention provides, for example, a novel microarray that can be used for expression analysis of abnormal proteins such as cancer and gene expression analysis, and a method for analyzing protein or gene expression using the same. Although 150 mg of tissue or cells was required as a specimen, the method of the present invention required much less than that. Particularly, according to the method of covering the upper surface with a cover glass, the specimen amount was about 2 mg or less. In this case, the required sample amount of the DNA or protein antibody array can be reduced.
That is, it is considered that analysis can be performed with a small amount of protein or RNA, and the expression analysis of protein or gene is expected to be greatly advanced.
The microarray of the present invention and the method of analyzing protein or gene expression using the microarray are extremely large in that they contribute to the study of protein expression analysis of cancer and the like, and to the study of disease mechanisms and therapeutic methods.
[Brief description of the drawings]
FIG. 1 shows an example of the microarray of the present invention. FIG. 1 (A) is a top view, FIG. 1 (B) is a side view (cross-sectional view), and FIG. 1 (C) is a sample solution. FIG.
FIGS. 2A and 2B show a microarray of the present invention in which an upper surface of an arrayed portion is covered with a cover glass or the like; FIG. 2A is a top view, and FIG. FIG. 2 (C) is a side view (cross-sectional view) in which a sample solution is placed.
3 shows the results of data analysis on the labeled protein mixture A-Cy3 / B-Cy5 obtained in Reference Example 1, wherein FIG. 3 (A) shows Cy3 and FIG. 3 (B) ) Shows the fluorescence of Cy5.
FIG. 4 shows the results of data analysis on the labeled protein mixture A-Cy3 / B-Cy5 obtained in Reference Example 1, and is a correlation diagram between A-Cy3 and B-Cy5. .
5 shows the results of data analysis on the labeled protein mixture B-Cy3 / A-Cy5 obtained in Reference Example 1, wherein FIG. 5 (A) shows Cy3 and FIG. 5 (B) ) Shows the fluorescence of Cy5.
FIG. 6 shows the results of data analysis on the labeled protein mixture B-Cy3 / A-Cy5 obtained in Reference Example 1, and is a correlation diagram between B-Cy3 and A-Cy5. .
7 shows the results of data analysis on the labeled protein mixture A-Cy3 / B-Cy5 obtained in Example 1, FIG. 7 (A) shows Cy3 and FIG. 7 (B) ) Shows the fluorescence of Cy5.
8 shows the results of data analysis on the labeled protein mixture A-Cy3 / B-Cy5 obtained in Example 2, FIG. 8 (A) shows Cy3 and FIG. 8 (B) ) Indicates the fluorescence of Cy5.
[Explanation of symbols]
1 slide glass
2 Part where antibody or DNA is arrayed
3 Hydrophobic sheet
4. Antibody or DNA spots
5 Sample inlet (gap)
6 Cover glass, etc.
7 Small space that can accommodate sample
8. Protein solution (sample solution)

Claims (19)

A microarray in which a hydrophobic sheet is attached to an outer peripheral portion (peripheral portion) of an arrayed portion on a slide glass on which an antibody or DNA is arrayed. The microarray according to claim 1, wherein the hydrophobic sheet is one or more plastic sheets, laminated paper, and / or silicone sheets. The microarray according to claim 1, wherein the hydrophobic sheet is an adhesive sheet or an adhesive tape. The microarray according to any one of claims 1 to 3, wherein the thickness of the attached hydrophobic sheet portion is 0.5 to 2 mm. A microarray in which a hydrophobic sheet is attached to an outer portion (peripheral portion) of an arrayed portion on a slide glass on which an antibody or DNA is arrayed, leaving a gap that can serve as a sample injection port. The microarray according to claim 5, wherein the hydrophobic sheet is one or more plastic sheets, laminated paper, and / or silicone sheets. The microarray according to claim 5, wherein the hydrophobic sheet is an adhesive sheet or an adhesive tape. The microarray according to any one of claims 5 to 7, wherein the thickness of the attached hydrophobic sheet portion is 0.05 to 0.5 mm. The microarray according to any one of claims 5 to 8, wherein at least the upper surface of a portion of the slide glass on which the antibody or DNA is arrayed is covered with a cover glass or the like. Expression of proteins or genes using a microarray in which a hydrophobic sheet is attached to an outer portion (peripheral portion) of an arrayed portion on a slide glass on which an antibody or DNA is arrayed. analysis method. The expression analysis method according to claim 10, wherein a microarray in which the hydrophobic sheet is one or more plastic sheets, laminated paper, and / or silicone sheet is used. The expression analysis method according to claim 10 or 11, wherein the hydrophobic sheet uses a microarray that is an adhesive sheet or an adhesive tape. The expression analysis method according to any one of claims 10 to 12, wherein a microarray having a hydrophobic sheet portion having a thickness of 0.5 to 2 mm is used. A microarray is used in which a hydrophobic sheet is attached to an outer peripheral portion (peripheral portion) of an arrayed portion on a slide glass on which an antibody or DNA is arrayed, leaving a gap that can serve as a sample injection port. A method for analyzing the expression of a protein or a gene, characterized in that: The expression analysis method according to claim 14, wherein a microarray in which the hydrophobic sheet is one or more plastic sheets, laminated paper, and / or silicone sheet is used. The expression analysis method according to claim 14 or 15, wherein the hydrophobic sheet uses a microarray that is an adhesive sheet or an adhesive tape. The expression analysis method according to any one of claims 14 to 16, wherein a microarray having a hydrophobic sheet portion having a thickness of 0.05 to 0.5 mm is used. The expression analysis method according to any one of claims 14 to 17, wherein a microarray in which at least an antibody or DNA arrayed on a slide glass has an upper surface covered with a cover glass or the like is used. The expression analysis method according to any one of claims 10 to 18, wherein the protein is an abnormal protein.

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