JP2001037487A - Method of detecting rna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of detecting an RNA by preparing a standard cDNA and a cDNA for measurement, respectively, from a standard RNA and a detection object RNA followed by addition of different kinds of adapters to these cDNAs and measuring the ratio between the amounts of amplified products obtained by mixing and amplifying these adducts. SOLUTION: By preparing a cDNA for measurement and a standard cDNA, respectively, by reverse-transcribing a detection object RNA and a standard RNA, adding different kinds of adapters comprising nucleotides or the like with different length to the each groups consisting of standard cDNAs with stepwise adjusted concentrations, on the other hand, adding other kinds of adapters different from the above adapters to the cDNA for measurement, mixing the resultant these adapter-added cDNAs to amplify the mixtures by the use of an adapter primer and a gene-peculiar primer, measuring the ratio between the amounts of the cDNA for measurement and the standard cDNA and detecting an RNA from the result of this measurement, the detection object RNA is detected with high precision. This method is useful in the detection or the like of a gene participating in a specific disease.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting RNA.

[0002]

2. Description of the Related Art Northern hybridization is generally performed to quantify the level of gene expression.
At the laboratory level, usually 5 pg of RNA can be detected. However, when the expression level of the gene is extremely small, 0.3 to 3 μg of mRNA is required.
Therefore, it is difficult to apply Northern hybridization when only a limited amount of sample is available (eg, a clinical specimen).

[0003] The polymerase chain reaction (PCR) is a technique capable of detecting DNA or RNA with the highest sensitivity as compared with other techniques. However, for the quantification of gene expression by PCR, a control experiment must be performed using a DNA fragment with the same amplification efficiency as the target molecule as a so-called "internal control" (internal standard). Therefore, the operation is complicated. In addition, quantitative PC
In order to perform R, it is necessary to create a calibration curve for each gene to be quantified, so it takes time to conduct research and diagnose genes.

[0004]

An object of the present invention is to provide a method for detecting RNA.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, added different types of adapters to the cDNA to be detected synthesized from RNA and the cDNA for preparing a standard curve. It was found that the detection target RNA could be detected with high precision by performing PCR, and the present invention was completed.

That is, the present invention provides the following steps: (a) the RNA to be detected and the standard RNA are reverse-transcribed to obtain a measurement cDNA and a standard cDNA, respectively; A different adapter is added to each group of cDNA, and (c) an adapter of a different type from the adapter is added to the cDNA for measurement, and (d) the adapter obtained by (b) and (c). (A) mixing and amplifying the additional cDNA;
This is a method for detecting RNA, comprising: measuring a quantitative ratio of a measurement cDNA to a standard cDNA; and (f) detecting RNA from the measurement result. Different types of adapters include those containing nucleotides of different lengths. Further, examples of amplification include those using an adapter primer and a gene-specific primer. Hereinafter, the present invention will be described in detail.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to RNA used as a control (RNA for preparing a standard curve, which is referred to as "standard RNA").
CDNA (referred to as "standard cDNA")
And the cDNA synthesized from the target RNA to be detected (referred to as “RNA to be detected”) and the cDNA to be actually measured (referred to as “measurement cDNA”) in the same reaction system For measurement against standard cDNA by amplifying with
Measure the quantitative ratio of cDNA, and use the measurement result as an index to determine RN
It is characterized by detecting A.

As described above, when gene expression is detected by PCR, a calibration curve is prepared in a reaction system separate from the target molecule, using a DNA fragment having the same amplification efficiency as the target molecule as an internal standard. Control experiments must be performed.
If this operation can be performed in a single reaction system containing the target molecule, the detection result of the detection target can be easily obtained with a small error. The present inventor has added different types of adapters to amplification DNA so that each DNA after amplification can be accurately and simply measured in a single in vitro reaction. Identifiable. Hereinafter, each step of the method of the present invention will be described.

(1) Preparation of RNA and cDNA RNAs to be detected (RNA to be detected) include RNAs from various organs. However, the type of RNA is not limited to these. The type of RNA to be detected may be one type, or two or more types, but preferably 1 to 5 types. When two or more kinds are used, they may be derived from different tissues or cells or from the same tissue or cells. For example, RNAs of the same species but at different times (for example, several days after birth, several weeks after, and several years after) can be selected as RNAs to be detected.

On the other hand, RNA for preparing a standard curve (standard RNA) is
RNA of a different type from the RNA to be detected
Is selected as appropriate according to the type of. Therefore, for example, when the RNA to be detected is derived from the cerebellar extract, the standard RNA can be derived from the whole brain (cerebrum, diencephalon, midbrain, and medulla) extract except the cerebellum.

A cDNA corresponding to each RNA is synthesized from the above-mentioned RNA to be detected and the standard RNA using a reverse transcriptase to obtain a measurement cDNA and a standard cDNA, respectively. Preparation of the measurement cDNA and the standard cDNA can be performed by any known technique. For example, a method of preparing poly (A) + RNA from cells or tissues of various organs and reacting with reverse transcriptase (Gubler, U and Hoffman, BJ, Gene, 25, 263-269
(1983); Okayama, Hand Berg, P., Mol.Cell.Biol.,
18, 5294 (1982)), using a commercially available kit (cDNA synthesis kit: Lifetech Oriental, etc.)
And the like.

The cDNA population after the above-mentioned cDNA synthesis contains not only a target gene used as a measurement or standard cDNA but also a cDNA that does not correspond to the target gene. Therefore, in the present invention, the target gene in the cDNA is amplified by an adapter addition reaction and a PCR reaction using specific primers (for example, an adapter primer and a gene-specific primer, etc.) described below, and the quantitative ratio is measured. You can be able to.

(2) Preparation of Adapter As described above, the present invention is characterized in that DNAs to which different types of adapters are added are measured at once in the same reaction system, that is, in the same reaction test tube. is there. Different types of adapters are designed so that when a certain DNA is amplified, the other DNA can be distinguished, and a double-stranded adapter that can be ligated to a double-stranded cDNA for measurement. Means oligonucleotide. The type of adapter is not limited as long as the amplified fragments can be distinguished from each other, and examples include those having different oligonucleotide lengths.

In the present invention, the number (type) of adapters that can be used in the same reaction system is not particularly limited, but is preferably up to 6 to 7 types. Therefore, the type of adapter to be added to the standard cDNA and the type of adapter to be added to the measurement cDNA can be appropriately selected within the range of the type of adapter to be used. For example, if you want to measure one type of cDNA,
There is only one type of adapter to be added to the DNA. On the other hand, since it is necessary to obtain at least two data to prepare a standard curve, the concentration of the standard cDNA is adjusted in at least two steps. Therefore, the type of adapter is the minimum
There are three types. When the preferred number of adapters (6 to 7 types) is used, the number of adapters to be added to the standard cDNA is set to 5 to 6 types (that is, the number of steps for adjusting the concentration of the standard cDNA is set to 5 to 6 levels). be able to.

As described above, when three types of cDNAs are to be measured, the adapter to be added to the measurement cDNA is 3
And the number of adapters to be added to the standard DNA is, for example, 3 or 4 (that is, the number of steps to be adjusted is 3
Or four steps). The adapter used in the present invention is a single-stranded sequence of a protruding portion that can anneal to the cohesive end of the cDNA (which can be obtained by treatment with a restriction enzyme) (referred to as a cohesive end sequence). And a sequence common to each adapter (called a common sequence). The cohesive end sequence, the spacer sequence, and the common sequence are linked in this order (the spacer sequence and the common sequence). The consensus sequence forms a double strand) (FIG. 1).

In FIG. 1A, the cohesive end sequence 2 can be appropriately designed depending on the type of restriction enzyme used when cleaving cDNA reversely transcribed from RNA to an appropriate length. In addition, the common sequence 1 and the spacer sequence 3 can be arbitrarily designed. The spacer sequence can have not only different oligonucleotide chain lengths but also different spacer oligonucleotide sequences for each adapter used. However, the shortest adapter may or may not include the spacer sequence 3.

In the present invention, the common sequence 1 has a minimum unit of 20 to 30 bases. Therefore, by changing the sequence of the spacer, adapters of various lengths can be produced (FIG. 1A). For example, when six types of adapters are used, the minimum length of the adapter is set to 20 to 30 bases, and nucleotides of an appropriate length (2 to 4 bases) are sequentially added thereto, while removing the cohesive end sequence. An adapter having 20 bases can be prepared as the adapter having the shortest sequence, and an adapter having 32 to 54 bases can be prepared as the adapter having the longest sequence excluding the cohesive terminal sequence. As a result, adapters having different lengths according to the length of the spacer sequence can be obtained, and thereby, PCR products described later can be distinguished. These adapters can be
For example, it can be obtained by chemical synthesis using a DNA synthesizer manufactured by Perkin-Elmer. The scope of the present invention is not limited by the number of adapters and the number of oligonucleotides described above.

(3) Addition of Adapter Next, different types of adapters are added to the respective cDNAs for measurement. Similarly, different types of adapters are added to the standard cDNA. In the present invention, the adapter can be excessively mixed with each sample so that the amount of the adapter added to the cDNA for measurement is not insufficient. In this case, in order to remove the adapter that is excessively mixed, that is, in order to be able to collect only the sample to which the adapter is added, one of a specific substance and a substance that specifically reacts with the specific substance ( For example, an antigen for an antibody, its substrate for an enzyme,
Biotin against streptavidin) in each sample
It is preferable to add to cDNA. If the other of the above substances is immobilized on the solid phase of the reaction vessel, the cDNA to which the adapter has been added can be recovered by a specific reaction between the above substances (FIG. 1B). FIG. 1B illustrates biotin (labeled “-b” at the end of the cDNA), streptavidin 7, and magnetic beads 8 to facilitate recovery.

However, when the amount of RNA as a raw material is large, R
It is thought that the relative difference between the amount of cDNA obtained by reverse transcription of NA and the free adapter is small. Therefore, in such a case, it is not necessary to remove the adapter, so that the above-mentioned specific substance need not be added. Adapter and measurement
Ligation with cDNA and subsequent steps are performed as cDNA for measurement.
Three (the DNA _1, DNA ₂ and DNA _3), by way of one as a standard cDNA described below (Figure 2). In DNA _1, DNA ₂ and DNA ₃ is a measurement cDNA, (A obtained by changing the length, respectively AP1, the AP2 and AP3) adapter adds, so that can be distinguished by differences in the length of the adapter To The measurement cDNA to which the adapter has been added is prepared in a 1-fold amount (FIG. 2A).

On the other hand, the concentration of the standard cDNA is adjusted stepwise, and different types of adapters are added to each concentration of DNA. For example, adapters AP4, AP5 and AP6 having different lengths are added to the standard cDNA prepared stepwise in 10 times, 3 times and 1 time in FIG. However, DNA concentration adjustment is not limited to 10: 3: 1 as described above,
The best conditions can be set to create a standard curve. Therefore, when there are three types of adapters, the concentration can be arbitrarily set, such as 1: 10: 100, 1: 5: 25, or the like. In addition, “1 time amount” refers to a reference amount when the concentration is adjusted stepwise.

(4) Mixing and Amplification Each cDNA prepared as described above is mixed in one reaction tube, and an amplification reaction (PCR) is performed using the cDNA as a template and an adapter primer and a gene-specific primer. The adapter primer means an oligonucleotide having a sequence complementary to the sequence of the adapter used in the present invention and capable of hybridizing (FIG. 1).
C). However, it is necessary that at least 15 bases of the sequence of the adapter primer hybridize with the common sequence. In FIG. 1C, the nucleotide sequence of the adapter primer 5 can be arbitrarily designed and synthesized according to the sequence of the common sequence 1 in the adapter. in this case,
The length of the nucleotide sequence of the adapter primer 5 is 15 to 50 bases, preferably 25 to 30 bases. The adapter 5 to be hybridized is designed to hybridize with the longer strand of the adapter double strand.

In the present invention, it is preferable to label the adapter primer appropriately 9 so that the amplified product after PCR can be detected by the detector (FIG. 1C). Examples of the labeling substance of the label 9 include fluorescein and rhodamine in the case of a fluorescent label, and ³² P and ³⁵ S in the case of a radioactive substance label. Labeling of the labeling substance with the primer can be performed according to a known method.
On the other hand, the gene-specific primer means an oligonucleotide having a sequence complementary to the sequence of the measurement cDNA 4 and capable of hybridizing (FIG. 1C). Nucleotide sequence length of gene-specific primer 6 is 15-50
Bases, preferably 25-30 bases.

The PCR conditions can be appropriately changed depending on the cDNA to be amplified. For example, amplification conditions are
Denaturation step at 98 ° C for 15 seconds to 1 minute, preferably at 94 ° C for 20 seconds, 3
An annealing step at 7 ° C to 72 ° C for 15 seconds to 1 minute, preferably 50 ° C for 40 seconds, and 50 ° C to 75 ° C for 15 seconds to 1 minute, preferably 7 ° C
One cycle of the elongation process at 2 ° C for 40 seconds is 30-40
Cycles are performed, preferably 35 cycles. However, Taq Gold
When using (Perkin-Elmer), it is preferable to add an activation step at 95 ° C. for 10 minutes before the above amplification cycle, and to completely elongate the unextended fragment of the amplified DNA. Alternatively, an extension step at 72 ° C. for 10 to 30 minutes can be added after the amplification cycle. In addition, the amplification product can be stored frozen (eg, −20 ° C.) so that measurements can be taken on another day.

(5) Detection The obtained amplification product can be detected by various methods. For example, when a primer used for PCR is labeled with a fluorescent dye or the like in advance, it can be detected as fluorescence intensity, and when labeled with a radioactive substance, it can be detected with a densitometer after exposure to an X-ray film. Also,
Perform agarose gel electrophoresis, polyacrylamide gel electrophoresis, etc. on the amplification product, and add ethidium bromide, SYBR
It can be stained with Green solution or the like, and the intensity of the band of the amplification product can be measured.

When different types of adapters are ligated (added) to each group of standard cDNA whose concentration is adjusted stepwise,
In the measurement step after PCR, the standard cDNA to which the adapter is added is separately measured for each fragment in accordance with the type of the adapter. According to the method of the present invention, a standard curve (for example, a mathematical function such as a linear function or a quadratic function) is created based on each of the measured values of the standard cDNA, and the measurement of the measurement cDNA is performed on the standard curve. By extrapolating the values, the standard cD
Measure the quantitative ratio to NA.

[0026] The amplification product is analyzed using a predetermined measuring instrument (for example, Perkin
-Elmer's sequencer).
In FIG. 2, standard cDN prepared in 10-fold, 3-fold, and 1-fold amounts
Assuming that the measurement result (fluorescence intensity) of A appears as P1, P2, and P3 with the peak position being different due to the difference in the adapter chain length (FIG. 2B, black peak), the standard curve is shown in FIG. 2C. Can be expressed as The graph in Figure 2C is 1
By substituting the fluorescence intensity of the measurement cDNA into the equation of the function, the measurement cDNA can be expressed as the following function.
Can know how many times the standard cDNA is expressed. For example, in FIG. 2, if the fluorescence intensity value obtained for DNA ₂ is 500 (P5 in FIG. 2B), the magnification is 5 according to FIG. 2C. Therefore, the measurement cDNA
₂ indicates that the expression is 5-fold relative to the standard cDNA. If the absolute amount of the mRNA derived from the gene in the DNA is known, the absolute amount of the cDNA for measurement can be known, and the
If the absolute amount of NA is not known, the relative ratio of expression can be determined.

Further, the absolute amount of the standard cDNA may be known or unknown. If the absolute amount of the cDNA is known, the absolute amount of the measurement cDNA can be determined, and the standard cDNA
If you do not know the absolute amount of
The amount ratio of cDNA can be known. Finally, using the measurement result obtained as described above as an index, the expression level ratio of the RNA of the gene in the tissue used as the sample is detected.

2. Analysis of Gene Expression Pattern by the Method of the Present InventionThe method of the present invention makes it possible to accurately detect the RNA for detection.Therefore, when the expression pattern of the same type of gene is detected for each individual development stage, various Gene expression patterns can be analyzed. For example, when the expression level of a certain mouse gene is measured several days after birth, around 10 days after birth, and several weeks after birth, some genes increase in expression as they grow and some genes decrease in growth. Also,
Some expression levels are constant regardless of the stage of development. Therefore, genes can be classified into several groups according to the mode of these expression patterns. Further, by detecting the expression pattern of a healthy person and the expression pattern of a gene of a subject suffering from a certain disease, it is possible to determine which gene has been changed.

[0029]

The present invention will be described more specifically with reference to the following examples. However, the technical scope of the present invention is not limited to these examples. Example 1 ATP synthase coupling factor (ATP syntha
In this example, the RNA of the ATP synthase coupling factor gene was detected.
The purpose of this is to detect. RNA from the target mouse cerebellar tissue was extracted with trizole (Giboco-BRL) or the like. 3 μg of total RNA and 1.5 pmol of 5 ′ biotinylated oligo dT were dissolved in 11 μl of distilled water. Next, 70 ° C 5
After heating for minutes, the temperature was returned to room temperature. Next, cDNA was synthesized from RNA.

(1) Synthesis of first-strand cDNA Reaction composition for cDNA synthesis 0.1 M DTT 2 μl 5x First-strand buffer (Gibco-BRL) 4 μl 10 mM dNTP 1 μl RNase inhibitor 1 μl

After heating the above reaction composition at 42 ° C. for 2 minutes,
1 μl of 200 units / μl of erscript II was added. 30 at 37 ° C
After heating for 40 minutes, the mixture was heated at 42 ° C. for 30 minutes. After transferring on ice,
The following second strand cDNA synthesis reagents were added.

(2) Synthesis of second strand cDNA Reaction composition for cDNA synthesis 10x Second strand synthesis buffer 10μl 10mM dNTP 4μl 100mM DTT 2μl E. coli DNA ligase 2μl (20 units) E.coi DNA polymerase I 4μl (40 units) E. coli RNase H 1μl ( 2 units) Total volume (adjusted with distilled water) 100 μl

After keeping the above reaction solution at 16 ° C. for 2 hours,
The enzyme was inactivated by heating at 30 ° C. for 30 minutes. 40 μl of the above reaction solution was transferred to a new tube.

(3) Cleavage by restriction enzyme Reaction solution composition 10x K buffer 10 μl cDNA solution above 40 μl Mbo I 10 units whole amount (adjusted with distilled water) 100 μl

The cDNA was cleaved by treating the above reaction solution at 37 ° C. for 30 minutes. Next, the cDNA fragment was extracted by phenol-chloroform extraction, and ethanol precipitation was performed. Rinse the cDNA twice with 70% ethanol, 5
It was dissolved in 00 µl of 0.1xTE buffer.

(4) Adapter addition reaction Reaction solution composition 10x T4 ligation buffer 7.5μl 10 pmol / μl MA-1L adapter 7.5μl 10 pmol / μl MA-1S adapter 7.5μl T4 DNA ligase 350 units (TaKaRa) Mbo I digested cDNA above 50μl (adjusted with distilled water) 75μl

The “L” and “S” in the adapter names MA-1L and MA-1S mean the long and short chains of each adapter, respectively. The reaction was kept at 16 ° C. overnight. The oligonucleotide sequences of the adapter and the adapter primer are as follows.

[0038]

In the present example, a cDNA derived from the brain obtained by removing the cerebellum from the whole brain of an adult mouse was used as the standard cDNA. this
The adapter MA-6, MA-3 or MA-1 is bound to the cDNA,
Various kinds of adapter-added cDNAs were prepared. On the other hand, the cerebellum-derived cDNA of the mouse was used as a detection target,
MA-2 for cDNA, MA-4 for 12 day old cDNA, and 6 day old
MA-5 was ligated to the weekly cDNA. After the adapter addition reaction, 25 μl of 5M NaCl was added, and 20 μl of 10 mg / ml streptavidin-coated paramagnetic beads were added, followed by adsorption on ice.

Next, cDNAs derived from whole brain and cerebellum derived from cerebellum at 4 days, 12 days and 6 weeks after birth were prepared in the following amounts, and each was mixed. Of the standard cDNAs, the one with MA-6 ligated to one-fold cDNA, the one with MA-3 ligated to three-fold cDNA, and the MA-1 ligated to ten-fold cDNA Was used. The detection target was cDNA derived from the cerebellum at 4 days, 12 days and 6 weeks after birth.
Was used, and the amount was set to 1 times in each case.

[0041] Whole brain-derived cDNA (connecting MA-1) 30 μl Whole brain-derived cDNA (connecting MA-3) 9 μl Whole brain-derived cDNA (connecting MA-6) 3 μl 3μl Cerebellar-derived cDNA (MA-5 ligated) 12 days old 3μl 16-week-old cerebellar cDNA (MA-5 ligated) 3μl

After washing the beads with 120 μl of distilled water, 120 μl of distilled water was added. Dispense 2.1 μl aliquots into a 96-well microtiter plate and add 0.4 μl (10 pmol /
μl) of a gene-specific primer (specific primer for the ATP synthase coupling factor gene) sequence was added. Sequence of gene-specific primer (same for both cerebellum-derived and whole brain-derived cDNA): 5′-ATGACAAATTACCACATGGA-3 ′ (SEQ ID NO: 14) Next, 2.5 μl of a 2 × TaqGold mixture was added (5 μl / sample). The composition of the reaction solution is as follows.

[0043] 2x TaqGold mixture 10x PCR buffer 54 μl 20 mM dNTP 4 μl 10 pmol / μl 6-FAM- C1S 20 μl TaqGold 5 μl (25 units) Total volume (adjusted with distilled water) 270 μl

The PCR reaction was performed under the following conditions. That is, after reacting at 95 ° C for 10 minutes to activate TaqGold,
A cycle of 20 seconds at 50 ° C., 40 seconds at 50 ° C., and 40 seconds at 72 ° C. was defined as one cycle, and 40 cycles were performed. After the reaction is completed,
Keep warm for a minute. To the reaction solution was added 5 μl of a T4 mixture, and the mixture was kept at 37 ° C. for 2 hours.

[0045] Composition of T4 mixture 27μl 10x K bufferT4 DNA polymerase 20 units Total volume (adjusted with distilled water) 270μl

1 μl of the above T4 mixture was taken and 7 μl of formamide dye was added. An equal amount of the marker mix was added to the diluted sample, and heated at 95 ° C. for 3 minutes. After heating, add 0.8 μl of DNA sequencer (Perkin-Elmer
Company). As a result, the peak of the fluorescence intensity shown in FIG. 3a was obtained. In FIG. 3a, the black peak is for standard cDNA and the white peak is for mouse cerebellum-derived DNA. When a standard curve was drawn based on the fluorescence intensity of the black peak, the one shown in FIG. 3B was obtained. Based on the standard curve, the cDNAs of the mice at 4 days, 12 days, and 6 weeks after birth were measured.
It was found to have expression levels of 1, 4, and 2.8 times.

Example 2 In this example, the expression ratio was measured for each of the five types of genes in the same manner as in Example 1. The standard cDNA was a whole brain-derived cDNA as in Example 1.
It was used. Table 1 shows the results.

[0048]

[Table 1]

In Table 1, A is a mouse serine / threonine protein kinase DLK gene, B and E are unidentified novel genes, C is a mouse local adhesion kinase gene, and D is a mouse ribosomal protein S6 kinase gene (rsk). The results are shown. Among A, MA-2, MA-4 and MA-5 show the detection results (fluorescence intensity) when the adapters MA-2, MA-4 and MA-5 are added to the genes, respectively, for MA-1, MA- 3 and MA-6
Is the adapter MA-1 (10
2 shows the detection results (fluorescence intensity) when MA-3 (3 times) and MA-6 (1 time) were added. The correlation coefficients of the standard curves A to E are close to 1, indicating that the correct curves are drawn.

Based on the above results, the ratio of the expression level in each sample to the whole brain was calculated using a standard curve, and compared with the case where only the whole brain cDNA to which MA-3 was added as the standard cDNA was used. Was done. Table 2 shows the results. A to E
In, the upper numbers indicate the expression ratio obtained using the standard curve, and the lower numbers indicate one standard cDNA (MA-3 added whole brain cD
The ratio calculated using only N) is shown. As shown in Table 2, there is a large variation between the numerical values in the upper row and the numerical values in the lower row (Tables 2, A and D), and it was found that accurate measurement can be performed by creating a standard curve. .

[0051]

[Table 2]

Example 3 Classification of Genes 419 DNAs selected from a pool (about 7000) of nucleotide sequences of clones randomly extracted from a mouse cerebellum cDNA library were subjected to 4 days after birth (granular cell At its peak)
The relative amounts of expression in three kinds of specimens at 12 days after birth (at the peak of granule cell migration and axon elongation synapse formation) and at 6 weeks after birth (adult) were measured by the method of the present invention. The adapter used was the same as in Example 1, and a cDNA derived from the whole mouse brain (excluding the cerebellum) was used as the standard cDNA. Adapter ligation, PCR and detection were performed as in Example 1. Cluster analysis (Ward method) was performed to classify genes according to their expression patterns.

As a result, 419 genes were classified into 19 clusters. These clusters were divided into four large groups (FIG. 4). FIG. 5 shows the expression pattern of each cluster. In FIG. 5, a is a gene that increases with development, b is a gene that shows an almost constant expression level regardless of the expression stage, c is a gene that shows peak expression on the 12th day after birth, and d is an expression level that decreases with development. It is a gene expression pattern. The number assigned to each graph in FIG. 5 corresponds to the number of each cluster in FIG. Thus, for example, group 1 (clusters 1-4) of FIG.
Shows that the expression level increases with development as shown in FIG. 5a.

The above 419 genes are divided into four groups: a gene group encoding a protein kinase, a gene group encoding a ribosomal protein, a gene group encoding a skeletal muscle protein, and a gene group encoding a protein involved in brain. We divided them into categories and examined which groups these genes belonged to. FIG. 6 shows the results. For example, most ribosomal proteins belong to the group with the peak at 4 days after birth, and genes related to the cytoskeleton belong to groups 3 and 4 only. In contrast, protein kinases do not have a characteristic expression pattern. In addition, the cerebellum-specific gene was expressed in Group 3 with the highest expression level on the 12th.
Is found only in group 1 where the adult has the highest expression level. This indicates that the cerebellar-specific expression pattern and the expression pattern during development are closely linked.

[0055]

According to the present invention, a method for detecting RNA is provided. Since the method of the present invention can detect the expression level of a target gene with high accuracy, it is useful for detecting a gene involved in a specific disease.

[0056]

[Sequence List] SEQUENCE LISTING <110> KATO, Kikuya <120> A method for detecting RNA <130> P99-0599 <150> JP99 / 145440 <151> 1999-05-25 <160> 14 <170> PatentIn Ver. 2.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 1 gatccgcgtt ctaacgacaa tatgtac 27 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 2 gtacatattg tcgttagaac gcg 23 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 3 gatctcttag cgttctaacg acaatatgta c 31 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 4 gtacatattg tcgttagaac gctaaga 27 <210> 5 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 5 gatccacgat tagcgttcta acgacaatat gtac 34 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 6 gtacatattg tcgttagaac gctaatcgtg 30 <210> 7 <211> 37 <21 > DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 7 gatcgagcac tcttagcgtt ctaacgacaa tatgtac 37 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA < 400> 8 gtacatattg tcgttagaac gctaagagtg ctc 33 <210> 9 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 9 gatcctaagc taccagttag cgttctaacg acaatatgta c 41 <210> 10 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 10 gtacatattg tcgttagaac gctaactggt agcttag 37 <210> 11 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 11 gatcagcgtt agagccttta gtgcgttcta acgacaatat gtac 44 <210> 12 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 12 gtacatattg tcgttagaac gcactaaagg ctctaacgct 40 <210> <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 13 gtacatattg tcgttagaac gc 22 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 14 atgacaaatt accacatgga 20

[0057]

[Sequence List Free Text] SEQ ID NO: 1: Synthetic DNA SEQ ID NO: 2: Synthetic DNA SEQ ID NO: 3: Synthetic DNA SEQ ID NO: 5: Synthetic DNA SEQ ID NO: 6: Synthetic DNA SEQ ID NO: 7: Synthetic DNA SEQ ID NO: 8: Synthetic DNA SEQ ID NO: 9: Synthetic DNA SEQ ID NO: 10: Synthetic DNA SEQ ID NO: 11: Synthetic DNA SEQ ID NO: 13: Synthetic DNA SEQ ID NO: 14: Synthetic DNA

[Brief description of the drawings]

FIG. 1 is a schematic view of an adapter used in the present invention.

FIG. 2 is a diagram showing an outline of a detection method of the present invention.

FIG. 3 is a diagram showing the results of detecting the expression pattern of the mouse cerebellum.

FIG. 4 is a diagram showing a cluster analysis result.

FIG. 5 is a diagram showing expression patterns of respective clusters classified into four groups.

FIG. 6 is a diagram showing classification results of various genes contained in a postnatal cerebellum of a mouse.

[Explanation of symbols]

1: Common sequence 2: Cohesive end sequence 3: Spacer sequence 4: cDNA for measurement 5: Adapter primer 6: Gene-specific primer 7: Streptavidin 8: Magnetic beads
9: Sign

Claims (3)

[Claims] 1. The following steps: (a) The RNA to be detected and the standard RNA are reverse transcribed, respectively, to obtain a measurement cDNA and a standard cDNA, and (b) a group of standard cDNA whose concentration is adjusted stepwise. (C) adding a different type of adapter to the cDNA for measurement, and (d) the adapter-added cDNA obtained by the (b) and (c).
Mixing and amplifying; (e) measuring the quantitative ratio of the measurement cDNA to the standard cDNA; and (f) detecting RNA from the measurement result, wherein the R
NA detection method. 2. The detection method according to claim 1, wherein the different kinds of adapters are composed of nucleotides having different lengths. 3. The detection method according to claim 1, wherein the amplification is performed using an adapter primer and a gene-specific primer.