JP2008035801A - Method for estimating existence ratio of nucleic acid having sequence with high homology using microarray - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid detection method independent of probe design by accurately assaying a cross hybridization ratio between nucleic acids having sequences with a high homology and a method for calculating the existence ratio of nucleic acids having sequences with a high homology. <P>SOLUTION: The method for estimating the existence ratio of nucleic acids having sequences with a high homology comprises a process (1) for preparing probes having completely complementary sequences corresponding to each of nucleic acids having sequences with a high homology and making a microarray loading the probes, a process (2) for individually hybridizing the microarray with each of nucleic acids having sequences with a high homology, detecting hybrids and making a numerical expression for calculating the existence ratio of nucleic acids having sequences with a high homology and a process (3) for applying a specimen having an unknown existence ratio of nucleic acids having sequences with a high homology to the microarray, detecting hybrids and substituting the detected result for the numerical expression of the process (2) and calculating the existence ratio of nucleic acids having sequences with a high homology in the specimen. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to microarray data analysis.

  A microarray is obtained by immobilizing a large number of probes on a carrier at high density and independently so as not to mix with each other. The probe mounted on the microarray serves as a sensor for capturing a nucleic acid molecule consisting of a sequence complementary to the base sequence by hybridization.

  Known microarrays include a surface-processed carrier such as glass or silicon, or a gel carrier in which probes are immobilized. For example, using a substance with a protective group that is selectively removed by light irradiation, and combining photolithography and solid-phase synthesis techniques, DNA is selectively synthesized in a predetermined region (reaction site) of a minute matrix ( A microarray can be produced by masking. A microarray is prepared by arranging a solution containing probe DNA separately prepared in advance by PCR or artificial synthesis on a chip surface in a small volume of several nl to several pl with a spotter or an arrayer and fixing it on a specific region on the substrate. Can be produced.

  Furthermore, a microarray using a through-hole base is also known. For example, it can be produced by regularly arranging a plurality of hollow fibers in the fiber axis direction, fixing a probe to each hollow fiber, and then transversely cutting the hollow fiber array (Patent Document 1).

  Nucleic acid molecules can be detected using the above microarray by hybridizing a nucleic acid sample to be tested to a nucleic acid microarray in a sequence-specific manner, and after hybridization with individual probes and nucleic acid molecules that are sequence-specifically bound thereto. Hybridization is performed by detecting a signal with a fluorescent substance or the like. By this detection, nucleic acid molecules in a nucleic acid sample corresponding to a plurality of probes can be examined at a time. Therefore, it is widely used to analyze the expression level of a plurality of nucleobase sequences or the sequence of a specific nucleobase sequence itself.

  In conventional detection using a nucleic acid microarray, hybridization is carried out under appropriate conditions set in advance, and then a nucleic acid sample remaining on the surface of the nucleic acid microarray and other unnecessary substances are removed by a washing step, which is specific to the probe. A nucleic acid sample forming a hybrid is detected. The probe is designed to be complementary or identical to the desired nucleobase sequence to be detected, such as sequence analysis or functional analysis. As the probe, a relatively long-chain nucleic acid such as cDNA, a short-chain synthetic oligonucleic acid, or the like is used. When using synthetic oligonucleic acid as a probe, for organisms that have accumulated knowledge of genetic information, such as humans and mice, using their base sequence information, paying attention to the homology and function of each sequence, The sequence of the synthetic oligonucleic acid is designed and a probe is prepared.

However, when preparing a probe, there are cases where a plurality of highly homologous sequences exist in the living body as a base sequence to be detected. For example, in the case of a nucleic acid having a short sequence number such as a microRNA (miRNA) having a base chain length of about 20 bases, the difference in base sequence is often only about a few bases. In such a case, it becomes very difficult to design the probe while maintaining the specificity between the probe and the target nucleic acid sequence. Therefore, the detection result by hybridization may include not only perfect complementation but also a signal of a sequence differing by one base, for example, and cannot be directly applied as a detection result of the target nucleic acid molecule. The present invention aims to solve the above problems.
Patent No. 3510882

  As a result of intensive studies to solve the above problems, the present inventor obtained a plurality of types of probes having high homology by obtaining detection data in the following steps, thereby obtaining a plurality of probes included in unknown sample nucleic acids. The present invention has been completed by finding that the abundance ratio of nucleic acids having a highly homologous sequence of a kind can be estimated almost accurately.

  That is, the present invention is a method for estimating the abundance ratio of nucleic acids having a highly homologous sequence, including the following steps.

(1) A step of preparing a probe having a completely complementary sequence corresponding to each of nucleic acids having a highly homologous sequence and preparing a microarray on which those probes are mounted.

(2) Step of creating a mathematical formula capable of individually hybridizing each nucleic acid having a highly homologous sequence to the microarray, detecting a hybrid, and calculating the abundance ratio of nucleic acids having a highly homologous sequence .

(3) A sample having an unknown abundance of nucleic acids having a highly homologous sequence is applied to a microarray, a hybrid is detected, and the detection result is substituted into the mathematical formula of (2) above to obtain a highly homologous sequence in the sample. Calculating the abundance ratio of nucleic acids.

  The present invention makes it possible to accurately measure the abundance of highly homologous nucleic acids that could not be measured specifically and sequence-specifically in conventional microarray experiments. It was.

    The present invention is a method for estimating the abundance ratio of nucleic acids having highly homologous sequences, including the following steps.

(1) A step of preparing a probe having a completely complementary sequence corresponding to each of nucleic acids having a highly homologous sequence and preparing a microarray on which those probes are mounted.

(2) Step of creating a mathematical formula capable of individually hybridizing each nucleic acid having a highly homologous sequence to the microarray, detecting a hybrid, and calculating the abundance ratio of nucleic acids having a highly homologous sequence .

(3) A sample having an unknown abundance of nucleic acids having a highly homologous sequence is applied to a microarray, a hybrid is detected, and the detection result is substituted into the mathematical formula of (2) above to obtain a highly homologous sequence in the sample. Calculating the abundance ratio of nucleic acids.

  Hereafter, each process is demonstrated sequentially.

  First, in the first step, probes having completely complementary sequences corresponding to nucleic acids having highly homologous sequences are prepared, and a microarray on which these probes are mounted is prepared.

  A nucleic acid having a highly homologous sequence is a nucleic acid having a difference of about one base within at least 20 bases. For example, a variant derived from the same gene in mRNA, a homolog, the same family species of miRNA, a gene polymorphism Examples include molds. In addition, any nucleic acid may be used regardless of RNA or DNA as long as a slight difference is observed in the hybridization efficiency between perfect match and mismatch. First, probes having sequences completely complementary to these nucleic acids are designed, and a microarray on which they are mounted is prepared.

  A microarray means a device that can immobilize probes such as oligonucleotides and cDNAs in a plurality of compartments and can monitor gene expression patterns, screen for new genes, detect gene mutations, detect polymorphisms, and the like. Various forms of microarrays are known. For example, a microarray in which probes are fixed at high density on a substrate using an optical lithography technique, a microarray in which probes are fixed on a glass substrate by spotting, and the like are known. A fiber-type microarray is also known (see Japanese Patent No. 3510882).

  In the present invention, a microarray with a large amount of immobilized probes is used in order to reduce the bias derived from the difference between the probe and the analyte amount ratio in the process of creating simultaneous equations described later and the process of using an unknown sample. Preferably used. Moreover, since washing and detection are repeated, a microarray that can be detected without drying the surface of the microarray during detection is preferably used.

  As such a microarray, a microarray in which a plurality of through holes are present in the substrate and probes are fixed to the through holes via a gel is preferably used. Examples of such a microarray include the microarray described in JP-A-2000-60554 and a fiber type microarray.

  Hereinafter, the fiber type microarray will be described in more detail.

  The fiber type microarray can be produced, for example, by the following steps a) to d).

a) A step of producing an array by arranging a plurality of hollow fibers such that the longitudinal directions of the hollow fibers are the same.

b) A step of embedding the array body to produce a block body.

c) A step of introducing a gel precursor polymerizable solution containing a probe into the hollow part of each hollow fiber of the block body, carrying out a polymerization reaction, and holding the gel-like substance containing the probe in the hollow part.

d) A step of cutting the block body into pieces in a direction crossing the longitudinal direction of the hollow fiber.

  Examples of the hollow fiber material include polyamide-based hollow fibers such as nylon 6, nylon 66 and aromatic polyamide, polyester-based hollow fibers such as polyethylene terephthalate, polybutylene terephthalate, polylactic acid, polyglycolic acid and polycarbonate, and polyacrylonitrile. Acrylic hollow fibers, polyolefin hollow fibers such as polyethylene and polypropylene, polymethacrylate hollow fibers such as polymethyl methacrylate, polyvinyl alcohol hollow fibers, polyvinylidene chloride hollow fibers, polyvinyl chloride hollow fibers, polyurethane hollow Examples thereof include fibers, phenolic hollow fibers, fluorine hollow fibers made of polyvinylidene fluoride, polytetrafluoroethylene, and the like, polyalkylene paraoxybenzoate hollow fibers, and the like.

  The hollow fibers are arranged three-dimensionally so that their longitudinal directions are the same. As an arrangement method, for example, a plurality of hollow fibers are arranged in parallel at a predetermined interval on a sheet-like material such as a pressure-sensitive adhesive sheet, and the sheet is formed into a sheet, and then the sheet is wound up in a spiral shape (Japanese Patent Laid-Open No. Hei 11- No. 108928). Further, two porous plates each having a plurality of holes provided at a predetermined interval are overlapped so that the hole portions coincide with each other, the hollow fibers are passed through the hole portions, and the interval between the two porous plates is opened. There is a method of filling a curable resin raw material around the hollow fiber between two perforated plates and curing it (see JP 2001-133453 A).

  The array is then embedded so that the array is not disturbed. Examples of the embedding method include a method in which a polyurethane resin, an epoxy resin or the like is poured into a gap between fibers, and a method in which fibers are bonded together by heat fusion.

  Next, a gel precursor polymerizable solution containing a probe is introduced into the hollow part of each hollow fiber of the embedded array, and a polymerization reaction is performed in the hollow part. As a result, the gel is held in the hollow portion, and the probe is fixed to the gel.

  Examples of the gel precursor solution include acrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N-acryloylaminoethoxyethanol, N-acryloylaminopropanol, N-methylolacrylamide, N-vinylpyrrolidone, hydroxyethyl methacrylate, One or more monomers such as (meth) acrylic acid and allyl dextrin, and methylene bis (meth) acrylamide, polyethylene glycol di (meth) acrylate and the like as crosslinkable monomers are included. If the end of the probe is modified with an unsaturated functional group in advance, the probe is chemically bonded to the gel structure by copolymerizing with the gel precursor via the end of the probe (see JP 2004-163211 A). ).

  Next, the block body is cut into thin pieces by cutting in a direction crossing the longitudinal direction of the hollow fibers. The thin piece produced here can be used as a microarray. The thickness of the microarray is about 0.1 mm to 1 mm. The cutting can be performed by, for example, a microtome or a laser.

  In the above-described microarray, the probe is fixed to the hollow portion of the hollow fiber, that is, the gel in the through hole of the microarray. Therefore, since the probe is fixed to the three-dimensional structure, the probe is fixed in a certain space. This surrounding environment where the probe is fixed has a degree of spatial freedom, and different types of probes are fixed with a physical partition. Therefore, the above microarray differs from the microarray in which probes are fixed at a high density on a flat substrate, and the probe mounted thereon functions sufficiently as a probe, and further non-specific reaction with different types of adjacent probes. There is no. Therefore, the probe can be designed without the need to bind extra sequences that are not involved in hybridization, such as linkers and spacers, and can be immobilized on the microarray.

    In the second step, each nucleic acid having a highly homologous sequence is individually hybridized to the microarray prepared as described above, the hybrid is detected, and the nucleic acid having a highly homologous sequence is detected based on the detection data. Create a formula that can calculate the abundance ratio.

  Here, each of nucleic acids having a highly homologous sequence is individually hybridized to detect a hybrid, which means that nucleic acids A and B constituting a group having high homology, that is, high possibility of cross-hybridization exist. In this case, nucleic acid A and nucleic acid B are individually hybridized to the microarray, and the signal intensities of probe A and probe B when nucleic acid A is hybridized are detected. Next, nucleic acid B is similarly hybridized to a new microarray, and the signal intensity of probe A and probe B is detected.

  The data obtained in this way is converted into mathematical formulas. Specifically, the signal intensity of probe A when hybridizing nucleic acid A is A, the signal intensity of probe B is B ′, the signal intensity of probe A when hybridizing nucleic acid B is A ′, and the signal intensity of probe B Is B, the signal intensity J in the probe A and the signal intensity in the probe B of the nucleic acid A and nucleic acid B mixture hybridized under the same conditions are expressed by the following equations.

J = A x + A'y
K = B′x + B y
(Here, x and y are expressed as relative amounts of nucleic acid A and nucleic acid B.)
That is, the quantity ratio of the nucleic acid A and the nucleic acid B can be determined by obtaining the solutions x and y of the simultaneous equations.

  In the above description, two types of nucleic acids (nucleic acid A and nucleic acid B) have been described. However, the present invention can be similarly applied to the case where there are three or more types of nucleic acids.

  In many hybridizations, the hybridization efficiency changes in a sequence-dependent manner depending on the stringency at the time of washing, so the same microarray is washed several times while changing the stringency and detected each time. The number of simultaneous equations corresponding to the number of detections can also be created. Therefore, the more detection results with different stringency in the same microarray, the more objective the result.

  Next, in the third step, a specimen in which the abundance ratio of nucleic acids having a highly homologous sequence is unknown is applied to the microarray, hybrids are detected, and the detection result is substituted into the mathematical formula created in the second step. The abundance ratio of nucleic acids having a highly homologous sequence is calculated.

  Specifically, the detection result of the probe A of a sample whose nucleic acid having a highly homologous sequence is unknown is substituted for J in the above equation, the detection result of the probe B is substituted for K in the equation, and x And y can be calculated to calculate the abundance ratio of nucleic acid A and nucleic acid B.

  Hereinafter, the present invention will be described in more detail with reference to examples.

  As an example, a mathematical formula for estimating the abundance of oligo DNAs of mmu-miR-23a and mmu-miR-23b, which are mouse miRNA sequences, is prepared.

(1) Sample preparation
According to miRBase (http://microrna.sanger.ac.uk/) of Sanger Institute, the sequences of mmu-miR-23a and mmu-miR-23b are as follows.

mmu-miR-23a (SEQ ID NO: 1): aucacauugccagggauu u cc
mmu-miR-23b (SEQ ID NO: 2): aucacauugccagggauu a cc
Since these sequences have only one base difference, when a completely complementary probe is designed and mounted on a nucleic acid microarray, the target analytes cross-hybridize with each other's probes simply by hybridizing the specimens. Therefore, it is impossible to measure each quantitative ratio.

  First, the synthetic oligo DNA shown in Table 1 was synthesized by an automatic DNA synthesizer and prepared at a concentration of 1 μg / μl.

次に、表２に従い、検体Ａと検体Ｂを調製した。 [Table 1]

Next, according to Table 2, Sample A and Sample B were prepared.

調製した検体Ａ及び検体Ｂを、ULYSIS Alexa Fluor 647 Nucleic Acid Labeling Kit（Kreatech社製）を用いて、表３の通りに混合、標識し、標識化サンプルＬ−検体ＡとＬ−検体Ｂを作製した。Labeling buffer及び、ULS labeling reagentは、キットに添付のものを使用した。 [Table 2]

The prepared specimen A and specimen B are mixed and labeled as shown in Table 3 using ULYSIS Alexa Fluor 647 Nucleic Acid Labeling Kit (manufactured by Kreatech) to produce labeled samples L-sample A and L-sample B. did. Labeling buffer and ULS labeling reagent used were those attached to the kit.

Ｌ−検体Ａ及びＬ−検体Ｂを、８５℃で１５分間加熱し、その後氷上で急冷した。Ｌ−検体Ａ、Ｌ−検体Ｂを、Sephadex G25 column(Amersham Bioscience製)のプロトコールに従って精製した。およそ２５μｌの精製液から１μｌを新しいチューブに分取し、Milli-Q を９９９μｌ加えて、１０００μｌとした。希釈液から２０μｌの液を新しいチューブに分取し、Milli-Qを１００μｌ、２０×ＳＳＣ溶液を１５μｌ、２％ ＳＤＳ溶液を１５μｌ加え、Ｈ−Ｌ−検体Ａ、Ｈ−Ｌ−検体Ｂを作製した。 [Table 3]

L-Sample A and L-Sample B were heated at 85 ° C. for 15 minutes and then rapidly cooled on ice. L-Sample A and L-Sample B were purified according to the protocol of Sephadex G25 column (Amersham Bioscience). 1 μl from approximately 25 μl of the purified solution was dispensed into a new tube, and 999 μl of Milli-Q was added to make 1000 μl. Dispense 20 μl of the diluted solution into a new tube, add 100 μl of Milli-Q, 15 μl of 20 × SSC solution, and 15 μl of 2% SDS solution to prepare HL-Sample A and HL-Sample B. did.

(2) Preparation of microarray <Preparation of probe>
First, an oligonucleotide serving as a probe having the sequence shown in Table 4 was synthesized by an automatic DNA synthesizer.

mmu-miR-23a_asは、上述のmmu-miR-23a-tに対して完全相補配列であり、mmu-miR-23b_asは、上述のmmu-miR-23b-tに対して完全相補配列である。 [Table 4]

mmu-miR-23a_as is a completely complementary sequence to the above-mentioned mmu-miR-23a-t, and mmu-miR-23b_as is a completely complementary sequence to the above-described mmu-miR-23b-t.

In the final stage of synthesis, aminolink ^TM (manufactured by PE Biosystems) was reacted with the oligonucleotide and then subjected to deprotection, whereby an aminohexyl group was introduced at the end of each oligonucleotide. -O-aminohexyl oligonucleotide was prepared.

  Subsequently, these oligonucleotides were reacted with methacrylic anhydride to prepare 5'-terminal vinylated oligonucleotides (hereinafter referred to as probes).

<Manufacture of hollow fiber bundle flakes>
A hollow fiber bundle was manufactured using the array fixture shown in FIG. Note that x, y, and z in FIG. 1 are orthogonal three-dimensional axes, and the x-axis coincides with the longitudinal direction of the fiber.

  First, two perforated plates with a thickness of 0.1 mm were prepared in which holes having a diameter of 0.32 mm were provided with a total of 144 holes in 12 rows each in a vertical and horizontal direction, with the distance between the centers of the holes being 0.42 mm. These perforated plates were overlapped, and polycarbonate hollow fibers (added by 1% by mass of carbon black manufactured by Mitsubishi Engineering Plastics) were passed through all of the holes one by one.

  The position of the two perforated plates was moved in a state in which a tension of 0.1 N was applied to each fiber in the X-axis direction, and was fixed at two positions of 20 mm and 100 mm from one end of the hollow fiber. . That is, the interval between the two perforated plates was 80 mm.

  Next, the three surrounding surfaces of the space between the perforated plates were surrounded by a plate-like object. In this way, a container having an open top only was obtained.

  Next, the resin raw material was poured into the container from the upper part of the container. As resin, what added 2.5 mass% carbon black was used with respect to the total weight of a polyurethane resin adhesive (Nippon Polyurethane Industry Co., Ltd. Nippon Run 4276, Coronate 4403). The resin was cured by standing at 25 ° C. for 1 week. Next, the porous plate and the plate-like material were removed to obtain a hollow fiber bundle.

  During the production of the gel-filled hollow fiber array, a gel precursor polymerizable solution containing a monomer and an initiator mixed at a mass ratio shown in Table 5 was prepared.

次に、プローブを含むゲル前駆体重合性溶液をデシケーター内に設置した。デシケーター内を減圧状態にしたのち、中空繊維束の繊維束が固定されていない一方の端部をこの溶液中に浸漬した。デシケーター内に窒素ガスを封入し、中空繊維の中空部にゲル前駆体重合性溶液を導入した。次いで、容器内を７０℃とし、３時間かけて重合反応を行った。 [Table 5]

Next, the gel precursor polymerizable solution containing the probe was placed in a desiccator. After reducing the pressure inside the desiccator, one end of the hollow fiber bundle where the fiber bundle was not fixed was immersed in this solution. Nitrogen gas was sealed in the desiccator, and the gel precursor polymerizable solution was introduced into the hollow part of the hollow fiber. Subsequently, the inside of a container was 70 degreeC and the polymerization reaction was performed over 3 hours.

  In this way, a hollow fiber bundle was obtained in which the probe was held in the hollow part of the hollow fiber via the gel-like material.

  Next, the obtained hollow fiber bundle was sliced in a direction orthogonal to the longitudinal direction of the fiber using a microtome, and 50 thin sheet (DNA microarray) having a thickness of 0.5 mm were obtained.

(3) The HL -sample A and HL-sample B prepared in the hybridization procedure (1) are heated at 70 ° C. for 2 minutes, and then centrifuged at 10,000 rpm for 2 minutes for hybridization. Provided. Hybridization was performed for 16 hours at 50 ° C. under light shielding conditions. Washing was performed with 2 × SSC, 0.2% SDS, 50 ° C. for 40 minutes, and then with 2 × SSC, 50 ° C. for 10 minutes.

  For detection, each probe spot of the microarray after hybridization was imaged and digitized using a cooled CCD camera type fluorescence detection apparatus. The results are shown in Table 6.

次に、上記検出後のマイクロアレイを再度、２ｘＳＳＣ、５５℃で３０分間洗浄し、再検出に供した。得られた数値は、バックグラウンドシグナルを減算すると、表７の通りとなった。 [Table 6]

Next, the microarray after the detection was washed again at 2 × SSC and 55 ° C. for 30 minutes and subjected to redetection. The numerical values obtained were as shown in Table 7 when the background signal was subtracted.

（４）未知検体の存在比の推測方法
上記の実験結果より、mmu-miR-23a及びmmu-miR-23bの配列を含むが、存在比が不明な検体Ｃは、下記の連立方程式（式Ｉ）で、量比（x及びy）が推測できる。 [Table 7]

(4) Method for Estimating Abundance Ratio of Unknown Specimen From the above experimental results, specimen C, which contains the sequences of mmu-miR-23a and mmu-miR-23b, but whose abundance ratio is unknown, has the following simultaneous equations (formula I ) To estimate the quantity ratio (x and y).

検体のハイブリ結果を検出後、ＩとＪの値を上記の数式に当てはめることにより、ｘとｙの数値が算出される。このｘとｙの比率が、mmu-miR-23a-tとmmu-miR-23b-tの量比となる。 [Formula I]

After detecting the hybridization result of the sample, the values of x and y are calculated by applying the values of I and J to the above formula. The ratio of x and y is the quantitative ratio of mmu-miR-23a-t and mmu-miR-23b-t.

  In addition, the quantitative ratio of the specimen can be estimated by the following simultaneous equations (formula II).

検体のハイブリ結果を検出後、ＫとＬの値を上記の数式に当てはめることにより、ｘとｙの数値が算出される。このｘとｙの比率が、mmu-miR-23a-tとmmu-miR-23b-tの量比となる。 [Formula II]

After detecting the hybridization result of the specimen, the values of x and y are calculated by applying the values of K and L to the above formula. The ratio of x and y is the quantitative ratio of mmu-miR-23a-t and mmu-miR-23b-t.

(5) Demonstration of estimated values In order to confirm the consistency of the above simultaneous equations, measurement was performed using model samples (sample C and sample D). Sample C and Sample D were prepared according to Table 8.

調製した検体Ｃ及び検体Ｄを、上述の（１）と同様の方法で標識、精製し、Ｈ−Ｌ−検体Ｃ、Ｈ−Ｌ−検体Ｄを作製した。これらのサンプルは、mmu-miR-23a-tとmmu-miR-23b-tの量比が、１：３及び、１：１であるので、これらのサンプルを用いて得られる推定量比は、ｘ：ｙ＝１：３および、ｘ：ｙ＝１：１に近い値が得られるものと考えられる。 [Table 8]

The prepared specimen C and specimen D were labeled and purified by the same method as in (1) above, and HL-sample C and HL-sample D were prepared. Since these samples have a ratio of mmu-miR-23a-t to mmu-miR-23b-t of 1: 3 and 1: 1, the estimated ratio obtained using these samples is It is considered that values close to x: y = 1: 3 and x: y = 1: 1 are obtained.

  HL-sample C and HL-sample D were heated at 70 ° C. for 2 minutes, and then centrifuged at 10,000 rpm for 2 minutes, and subjected to hybridization. Hybridization was performed for 16 hours at 50 ° C. under light shielding conditions. Washing was performed with 2 × SSC, 0.2% SDS, 50 ° C. for 40 minutes, and then with 2 × SSC, 50 ° C. for 10 minutes.

  For detection, each probe spot of the microarray after hybridization was imaged and digitized using a cooled CCD camera type fluorescence detection apparatus. The results are shown in Table 9.

検出後、同一のマイクロアレイを再度、２ｘＳＳＣ、５５℃で３０分間洗浄し、再検出に供した。 [Table 9]

After detection, the same microarray was washed again at 2 × SSC at 55 ° C. for 30 minutes and subjected to re-detection.

The numerical values obtained were as shown in Table 10 when the background signal was subtracted.

表９及び表１０で得られた結果を式Ｉ及び式ＩＩの連立方程式のＩ、Ｊ、Ｋ及びＬに代入し、ｘ及びｙを求めた（表１１及び表１２）。 [Table 10]

The results obtained in Table 9 and Table 10 were substituted into I, J, K, and L of the simultaneous equations of Formula I and Formula II to obtain x and y (Tables 11 and 12).

[表１２]

即ち、H-L-検体Cにおいては、mmu-miR-23a-t：mmu-miR-23b-tは、５０℃洗浄時の推定値でおよそ１：３、５５℃洗浄時の推定値でおよそ１：３となった。 [Table 11]

[Table 12]

That is, in HL-specimen C, mmu-miR-23a-t: mmu-miR-23b-t is approximately 1: 3 when estimated at 50 ° C., and approximately 1: 3 when estimated at 55 ° C. It became 3.

  Moreover, in HL-specimen D, mmu-miR-23a-t: mmu-miR-23b-t is approximately 1: 1 at an estimated value at 50 ° C. washing, and approximately 1: at an estimated value at 55 ° C. washing. It became 1.

  These estimated values corresponded to the mixing ratio of mmu-miR-23a-t and mmu-miR-23b-t in HL-sample C and HL-sample D, 1: 3 and 1: 1 (Table 8). .

  Therefore, by using this method, the mixing ratio of mmu-miR-23a-t and mmu-miR-23b-t could be estimated accurately.

It is a figure which shows the jig for arranging a hollow fiber.

SEQ ID NO: 5: synthetic probe perfectly complementary to mmu-miR-23a SEQ ID NO: 6: synthetic probe perfectly complementary to mmu-miR-23b

Claims (3)

A method for estimating the abundance ratio of nucleic acids having a highly homologous sequence, comprising the following steps.
(1) A step of preparing a probe having a completely complementary sequence corresponding to each of nucleic acids having a highly homologous sequence and preparing a microarray on which those probes are mounted.
(2) Step of creating a mathematical formula capable of individually hybridizing each nucleic acid having a highly homologous sequence to the microarray, detecting a hybrid, and calculating the abundance ratio of nucleic acids having a highly homologous sequence .
(3) A sample having an unknown abundance of nucleic acids having a highly homologous sequence is applied to a microarray, a hybrid is detected, and the detection result is substituted into the mathematical formula of (2) above to obtain a highly homologous sequence in the sample. Calculating the abundance ratio of nucleic acids.   The method according to claim 1, wherein in the second step, hybrid detection is performed with different stringency, and a plurality of mathematical expressions are generated. The method according to claim 1 or 2, wherein the nucleic acid having a highly homologous sequence is composed of 10 bases or more and 30 bases or less.

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