JPH09234100A - Detection of nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply detect the existence of a target nucleic acid sequence in a specimen by transferring the specimen comprising a tissue, a cell, a subcellular biological structure, etc., to a thin film support, folding it thereon, and subsequently subjecting the formed sample to a hybridization method, etc. SOLUTION: This method for detecting the presence of a target sequence in a single-strand or double-strand nucleic acid in a specimen comprising a tissue, a cell, a subcellular biological structure, etc., comprises transferring and holding the specimen to a thin film support (e.g. a nitrocellulose film), holding it thereon, and subsequently subjecting the formed sample to a nucleic acid-proliferating method such as a southern and a northern hybridization methods or a polymerase chain reaction(PCR) method. Thus, the peeling of a sample slice, etc., on the preparation of a sample and on the detective operation of a gene can be prevented, and the existence of a target nucleic acid sequence in the sample can simply be detected.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for detecting a nucleic acid, and more particularly to a method for detecting a target sequence present in a single-stranded or double-stranded nucleic acid in a tissue or cell structure.

[0002]

2. Description of the Related Art Recent rapid advances in gene cloning technology have contributed to the isolation and analysis of numerous genes, and have brought important biological and medical results. In order to elucidate the function and expression control mechanism of the gene thus obtained, a method for studying the expression state of the gene at the tissue or cell level has also been developed at present. In situ hybridization method (I
SH method) is a sample consisting of, for example, a tissue, a cell, or a subcellular structure from a specimen biological material immobilized on a solid support in a state where the permeability of the membrane is increased while maintaining the basic structure. DNA or RNA contained in the DNA and DNA or RNA containing the nucleotide sequence of the target gene to be investigated
This is a method for detecting the presence of a target gene by hybridization with a probe (see, for example, Japanese Patent Publication No. 4-5
02553). It is also possible to detect the presence of the target gene in the above sample by a nucleic acid amplification method using a primer specific to the target gene, for example, PCR method, which is called in situ PCR method. (See Japanese Patent Publication No. 8-73). These methods are described in Cell Engineering Annex 9, Deisotope Experiment Protocol, DI.
G Hybridization, Chapters 7 and 9 (19
(Published by Shujunsha Co., Ltd. on March 25, 1994). These methods can obtain information on the presence or expression state of a gene at the tissue or cell level, and are widely used in the fields of biology and medicine.

[0003]

Problems to be Solved by the Invention When detecting a gene by the ISH method and the in situ PCR method,
The sample biological material of interest is held on a support, and is immobilized in a state in which the structure at the time of survival is left as a sample. Usually, a slide glass is used for this support because it is easy to observe the sample during detection.
In this case, the sample preparation requires complicated operations. Further, when a tissue section is used as a sample, there is a problem that the section is likely to be peeled off at the time of preparing the sample and at the time of gene detection operation, and the risk is high especially in a decalcified specimen often handled in the dentistry area. Further, since peeling of the section is likely to occur when the sample is processed at a high temperature, it is generally not desirable to process the sample at a high temperature (for example, 65 ° C. or higher). On the other hand, however, insufficient heat treatment causes a decrease in detection sensitivity due to insufficient denaturation of nucleic acid in the sample and an increase in background due to nonspecific annealing of the probe or primer with the nucleic acid in the sample. Invite. Thus, the currently used ISH
The method and the in situ PCR method still have problems to be solved.

[0004]

[Means for Solving the Problems] Recently, as one of the immunohistochemical methods for proteins, the freeze transfer method (McGrath eta)
l, Biotechniques, Vol. 11, pp. 352-361,
1991) has been developed. In this method, the frozen slices are thawed on a nitrocellulose membrane, the proteins in the slices are transferred onto the membrane, and then the proteins are detected immunochemically. This method has the advantage that the target protein can be detected with high sensitivity because the membrane has a large allowable amount of protein adsorption and a sample containing undenatured protein can be obtained. In addition, ordinary histochemical staining is also possible, and observation with an optical microscope can be performed by subjecting the film to a transparent treatment. Although this method is intended for proteins, the present inventors have noted that nucleic acids (DNA, RNA) also strongly adsorb to nitrocellulose membranes as shown in the examples of Southern and Northern hybridization methods. , We believe that it is possible to develop a new ISH method and in-situ PCR method that overcomes the conventional problems by using a sample held on a thin film support such as a nitrocellulose membrane. As a result, by using the thin film support and the freeze transfer method, a sample applicable to the ISH method and the in situ PCR method can be prepared by a simpler operation than the conventional method, and the obtained experimental results are It was found that the method was superior to the method, and the present invention was completed.

That is, the present invention provides a method for detecting a nucleic acid, which is characterized in that, when detecting the presence of a target sequence in a nucleic acid in a sample, the sample is prepared by transferring and holding the sample on a thin film support. It is a thing. Also, the present invention
Kits for use in such methods are also provided.

The method for detecting a nucleic acid of the present invention comprises preparing a sample by transferring a tissue section, cell or subcellular biological structure of a sample to a thin film support, preferably by applying the freeze transfer method. It is characterized by detecting the presence of the target sequence in the sample on the thin film, preferably by the ISH method or the in situ PCR method. Thereby, the method of the present invention eliminates the drawbacks of the conventional method, and enables the detection of nucleic acids and fragments thereof with higher sensitivity by a simpler operation than the conventional method,
Not only in the field of basic research, but also in the fields of clinical medicine and dentistry, it can be widely applied to the detection of nucleic acids.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Samples to which the method of the present invention is applied include
It is not particularly limited, and includes various biological materials such as cells and cells in the fields of basic research, clinical medicine, dentistry, etc., including polynucleotides such as single-stranded or double-stranded nucleic acids. . For example, in the basic medicine, when examining the mechanism of skeletal formation in the method of the present invention, the type I collagen gene, the type II collagen gene, the type IX collagen gene, the type X collagen gene, in a developing animal fetus sample, It can be used to study the expression of various matrix (extracellular matrix) genes such as aggrecan gene, and in clinical medicine, various disease markers and tumor markers in biopsy materials, and gene expression sites and expression of oncogenes. It can be used to study the level (quantity).

As the thin film support, a plastic polymer thin film having an affinity for a polynucleotide, for example, a nitrocellulose film, or a polymer film having the same property as that, for example, a nylon film or PVDF (polyvinylidene) is used. Difluoride) membranes and the like can be mentioned, and above all, a nitrocellulose membrane used in Southern or Northern hybridization method or Western blotting method can be preferably used. In addition, whether or not these polymer membranes are suitable for use in the method of the present invention can be determined by examining their ability to adsorb nucleic acids and proteins by a method known per se such as the dot blot method. You can look it up. By using the thin film support, the sample tissue or the like can be cross-linked to the support, and peeling of the sample from the support and loss of sample components can be prevented. In addition, compared to the slide glass used in the conventional ISH method, the retention amount of the specimen sample can be remarkably increased, and it becomes possible to retain a trace amount of mRNA, and the immobilization of ribonuclease causes the degradation of RNA during hybridization. Is less likely to occur, retention of large slices is improved, sample preparation becomes easier, and highly sensitive detection becomes possible.

In the preparation of the sample, the transfer and retention of the sample tissue or the like to the thin film support are carried out by capillary blotting using the capillary phenomenon (Tissue Printing, pp. 139-
151, 1992, Academic Press), but in the present invention, in particular,
The freeze transfer method is preferably used. For example, a thin film support cut into an appropriate size may be used for about 10 minutes and about 9
It is immersed in distilled water or salt solution (eg, 0.1M CaCl ₂ solution) at 0 ° C., air-dried, and fixed on a slide glass with a clip or the like. Then, the frozen specimen section is sufficiently dried on a thin film support with a dryer or the like. If desired
It may be UV-crosslinked by a method known per se. Then fix it. Next, enzyme treatment (eg, proteinase treatment, etc.), washing, and if necessary re-fixation treatment (eg, 4%)
Post-treatment such as paraformaldehyde treatment), dry with a drier, etc., and perform detection operation.

To detect the presence of the target sequence in the sample, it is possible to use a hybridization method by a method known per se or a nucleic acid amplification method represented by a PCR method.
As the PCR method, an in situ PCR method is preferable. It should be noted that non-specific reaction is eliminated by carrying out Northern hybridization in which the material obtained from the same sample is transferred to a nitrocellulose membrane to detect the target nucleic acid in parallel under the same conditions as the hybridization of the present invention. The conditions to do can be easily found. Moreover, in the in-situ PCR method, mRN amplified on a thin film support was used.
Since the diffusion of A is minimized, high efficiency and detection sensitivity can be obtained, and furthermore, it is possible to detect a gene that is expressed only in a trace amount.

The RNA probe for hybridization can be prepared by a method known per se. For example, a digoxigenin (DIG) -labeled RNA probe for detecting the type II collagen gene can be prepared as follows. It was made. J. Biol. Chem., Vol. 259, p
p. 13668-13673 (1989), encoding the N-terminal portion of rat α1 (II) collagen.
A plasmid containing DNA, pRcol2, was prepared and used as Pst
A recombinant plasmid p1377 (α1II) was prepared by introducing a DNA fragment of about 550 bp obtained by digesting with I into a plasmid vector pGEM (manufactured by Promega). 20 μl reaction mixture containing 1 μg of this plasmid as a template [4 μl
5 times concentrated RNA polymerase buffer, 2 μl of dithiothreil (DDT), 1 μl of ribonuclease inhibitor (40 U / μl, Promega), 2 μl of D
IG RNA Labeling Mixture (Boehringer
Mannheim) 1 μl RNA polymerase (20
μg / μl, manufactured by Promega Co.) was prepared. RNA
The polymerase is a transcription product of RNA on the antisense side.
To obtain T7 RNA polymerase and SP6
The plasmid used as a template was selected from RNA polymerases and used as a linear plasmid by cutting with a suitable restriction enzyme between the inserted DNA fragment and the promoter sequence on the side not recognized by the RNA polymerase used. .
After incubating this reaction solution at 37 ° C. for 60 minutes, 2 μl DDT, 1 μl ribonuclease inhibitor, 1 μl ribonuclease free
Deoxyribonuclease (50 U / μl, manufactured by Promega) was added, and sterile distilled water was further added to bring the total volume to 50 μl.
And This reaction solution was incubated at 37 ° C for 15 minutes to decompose the template DNA in the reaction solution, and then ethanol precipitation (-80 ° C, 60 minutes) was performed to recover the synthesized DIG-labeled RNA probe, and 50 µl of diethyl was added. A probe solution was prepared by dissolving in pyrocarbonate (DEPC) -treated distilled water.

Probes for detecting the genes of type I collagen, type IX collagen, type X collagen, and aggrecan are based on the genes described in the following literatures:
It can be prepared by performing the same operation as above. Type I collagen: (α1 (I) collagen cDNA): B
iochemistry, Vol. 23, pp. 6210-6216 (1
984), type IX collagen (α1 (IX) collagen cDN
A): J. Bone Miner. Res., Vol. 8, pp. 1377-
1387 (1993), type X collagen (α1 (X) collagen NC1 domain
cDNA): Dev. Biol., Vol. 170, pp. 387-3
96 (1995), aggrecan (plasmid pRC11 inserted DNA fragment):
J. Biol. Chem., Vol. 262, pp. 17757-17
767 (1987).

The PCR primer can be arbitrarily designed and used based on the nucleotide sequence of the gene to be detected so that the sequence or a part thereof can be amplified. There is no particular limitation in the design, but the template DNA
Consider general problems in primer design, such as the melting temperature (Tm value) between the primer and the primer becomes appropriate temperature, and the primer itself does not form a secondary structure in the molecule. Is desirable.

Thus, the method of the present invention can be carried out, for example, in the following steps. Method 1 (1) Cut the thin film support into an appropriate size. 90 support
Immerse in distilled water at 0 ° C. or 0.1 M CaCl ₂ solution for 10 minutes, air dry, and fix the support to the slide glass with clips at the time of use. (2) Place a frozen section of the sample on the support. Dry completely with a dryer. (3) Wash with buffer. (4) Treat with proteinase K for 10 to 60 minutes at room temperature (pH 8.0). (5) Immobilize by treating with 4% paraformaldehyde (pH 7.2) in buffer for 20 minutes at 4 ° C. (6) Treat with 0.2N HCl for 10 minutes at room temperature. (7) Wash with buffer. (8) At room temperature, 0.1M triethanolamine (pH)
Shake treatment for 10 minutes in 8.0). (9) At room temperature, 0.1M triethanolamine (pH)
8.0), treated with 0.25% acetic anhydride for 10 minutes at room temperature. (10) Wash with buffer. (11) Prepare a sample by drying with a dryer or the like. (12) This support is cut into an appropriate size and placed in a tube (Eppendorf tube or the like) or a sealable multi-well plate or the like.

(13) For example, prehybridization is performed at 65 ° C. for 3 hours or more. Hybridization buffer: 50% formamide, 10 mM Tris-H
Cl (pH 7.6), 1 mM EDTA, 0.2% SDS, 1
× Denhardt solution, 0.6M NaCl, 10
% Dextran sulfate, 200 μg / ml yeast t
RNA. (14) For example, regarding a DIG-labeled RNA probe,
Hybridization is performed overnight at 65 ° C. (15) 2 × SSC containing 50% formamide [1 × S
CC is washed with 0.15 M NaCl, 0.015 M sodium citrate (pH 7.0)] for 20 minutes, and the operation is repeated twice. (16) 10 mM Tris-HCl (pH 7.6), 500 m
Treat with MNaCl and 1mM EDTA (TNE) at 37 ° C for 10 minutes. (17) 10-40 μg of ribonuclease A in TNE
/ Ml for 10 to 30 minutes at 37 ° C. (18) Treat with TNE at 37 ° C. for 5 minutes. (19) Treat with 2 × SSC at 60 ° C. for 20 minutes. (20) Repeat the treatment with 0.2 × SSC at 60 to 65 ° C. for 15 minutes twice. (21) Dry with a dryer or the like. (22) D with alkaline phosphatase-conjugated anti-DIG antibody
IG labeled probe is detected.

Of these, steps (3), (6), (7),
(8), (9) and (16) to (19) can be omitted, and the following simplified method 2 can be adopted. Method 2 (1) Cut the thin film support into an appropriate size. 90 support
Immerse in distilled water at 0 ° C. or 0.1 M CaCl ₂ solution for 10 minutes, air dry, and fix the support to the slide glass with clips at the time of use. (2) Place a frozen section of the sample on the support. Dry completely with a dryer. (3) Treat with proteinase K for 10 to 60 minutes at room temperature (pH 8.0). (4) Immobilize by treating with 4% paraformaldehyde (pH 7.2) in a buffer solution at 4 ° C. for 10 to 20 minutes. (5) Wash with buffer. (6) Prepare a sample by drying with a dryer or the like. (7) This support is cut into an appropriate size and placed in a tube (Eppendorf tube or the like) or a sealable multi-well plate or the like.

(8) For example, prehybridization is performed at 65 ° C. for 3 hours or more. Hybridization buffer: 50% formamide, 10 mM Tris-HC
l (pH 7.6), 1 mM EDTA, 0.2% SDS, 1 ×
Denhardt solution, 0.6M NaCl, 10%
Dextran sulfate, 200 μg / ml yeast tR
NA. (9) For example, regarding the DIG-labeled RNA probe, 6
Hybridization is performed overnight at 5 ° C. (10) 2 × SSC, then 60% with 50% formamide
The operation of washing at ˜65 ° C. for 15 to 20 minutes is repeated twice. (11) Repeat the treatment with 0.2 × SSC at 60 to 65 ° C. for 15 minutes twice. (12) Dry with a dryer or the like. (13) D with alkaline phosphatase-conjugated anti-DIG antibody
IG labeled probe is detected.

On the other hand, the conventional ISH method has the following steps.
For example, the following method 3 is used, and all the steps cannot be omitted. Method 3 (1) Wash the slide glass thoroughly with a neutral detergent (for about 60 minutes). After washing with water for 10 minutes, it is autoclaved at 120 ° C. for 20 minutes, further washed with water for 10 minutes, and then dried at 180 ° C. for 3 hours. Then in acetone,
Immerse in 2% aminopropyltriethoxysilane for 10 seconds and completely dry with a dryer or the like. (2) Place the frozen section of the sample on the slide glass. Dry completely with a dryer. (3) Immobilize by treating with 4% paraformaldehyde (pH 7.2) in buffer for 20 minutes at 4 ° C. (4) Wash with buffer. (5) Treat with proteinase K for 10 to 60 minutes at room temperature (pH 8.0). (6) Wash with buffer. (7) Immobilize by treating with 4% paraformaldehyde (pH 7.2) in buffer for 5 minutes at 4 ° C. (8) Wash with buffer. (9) Treat with 0.2N HCl for 10 minutes at room temperature. (10) Wash with buffer. (11) At room temperature, 0.1M triethanolamine (pH)
Shake treatment for 10 minutes in 8.0). (12) At room temperature, 0.1M triethanolamine (pH)
8.0), treated with 0.25% acetic anhydride for 10 minutes at room temperature. (13) Wash with buffer. (14) Prepare a sample by drying with a dryer or the like.

(15) An appropriate amount of a hybridization buffer solution is dropped on the section of the slide glass holding the section, and the section is covered with parafilm or the like, and prehybridization is performed at 42 to 50 ° C. for 3 hours or more. . Hybridization buffer: 50% formamide, 10 m
M Tris-HCl (pH 7.6), 1 mM EDTA, 0.
2% SDS, 1 × Denhardt solution, 0.6
M NaCl, 10% dextran sulfate, 2
00 μg / ml yeast tRNA. (16) For example, regarding a DIG-labeled RNA probe,
Hybridization is performed overnight at 42-50 ° C. (17) 50 ° C. in 2 × SSC containing 50% formamide
The operation of washing for 20 minutes is repeated twice. (18) 10 mM Tris-HCl (pH 7.6), 500 m
Treat with MNaCl and 1mM EDTA (TNE) at 37 ° C for 10 minutes. (19) 10-40 μg of ribonuclease A in TNE
/ Ml for 10 to 30 minutes at 37 ° C. (20) Treat with TNE at 37 ° C. for 5 minutes. (21) Treat with 2 × SSC at 50 ° C. for 20 minutes. (22) Repeat the treatment with 0.2 × SSC at 50 ° C. for 15 minutes twice. (23) Dry with a dryer or the like. (24) D with alkaline phosphatase-conjugated anti-DIG antibody
IG labeled probe is detected.

When the detection is carried out by the nucleic acid amplification method, the nucleic acid amplification method is applied to the sample prepared as described above. For example, as the nucleic acid amplification method used in the present invention,
In addition to the PCR method, an amplification method using RNA polymerase (JP-A-2-5864) and a strand displacement amplification method (JP-B-7)
No. 114718) can be used. In the nucleic acid amplification method, for example, a primer used for amplification is labeled, or a labeled nucleotide triphosphate is added to the reaction solution to carry out an amplification reaction, and then amplified by a method according to the labeling compound used. Nucleic acid can be detected.

The present invention also provides a kit for detecting the presence of a target sequence by the above method. The kit may be any as long as it contains the thin film support used in the method of the present invention as an essential member, and for example, depending on the type of sample, the purpose of detection, etc. Fixing reagent used, hybridization reagent,
It may be combined with at least one reagent selected from PCR reagents, detection reagents and the like. Further, these reagents may be solid or liquid.

[0022]

EXAMPLES Next, the present invention will be described in more detail with reference to test examples and examples, but the present invention is not limited to these examples. Test Example 1 Comparison of Binding Properties of Specimen Sample to Poly L-Lysine Coated Slide Glass, Silane Coated Slide Glass, and Nitrocellulose Membrane In the conventional ISH method, poly L-lysine coated ( Huang, WH,
et al, (1983) Histochemistry, 77, 275-
279) or silane coat (Burns et al, (198
7) J. Clin. Pathol., 40, 858-864) is widely used. The binding properties of the specimen samples to the slide glass coated with these and the nitrocellulose membrane were compared.

Frozen sections of hind limbs of a mouse 14-day-old fetus (5
μm thickness) on a poly-L-lysine-coated slide glass, a silane-coated slide glass, or a nitrocellulose membrane (BA85-PH75 nitrocellulose membrane, manufactured by S & S Co., Ltd.) having a pore size of 0.05 μm, and dried with a dryer to make sections. Was transcribed and then incubated at 55 ° C., 65 ° C. and 75 ° C. in 50% formamide, 2 × SSC for 12 hours. Then, nuclear section
The samples (10 sections each) were stained with Fast Red and examined for the peeling state. The results are shown in Table 1. The results show that peeling (complete peeling or 70%
The number of peeled pieces is shown.

[0024]

[Table 1]

As shown in Table 1, it was found that the nitrocellulose membrane was far better in retaining the slices than the slide glass, and that the hybridization could be performed at a higher temperature.

Test Example 2 Examination of Simplification of ISH Method In the conventional ISH method, in order to reduce the background signal due to non-specific adsorption of the probe, as shown in Method 3 above, the section was treated with HCl (see above). , Cell Engineering Supplement 9, Chapter 7), acetylation (Hayashi et a
l, (1978) J. Histochem., Cytochem., 26
(8), pp. 677-679) and ribonuclease treatment after hybridization (Takabayashi et al,
(1995) J. Cell Biol., 129 (5), pp. 14
11-1419) and so on. On the other hand, after the RNA is electrophoresed, it is transferred to a nitrocellulose membrane or a nylon membrane and hybridized with a probe. In the Northern hybridization method in which the target mRNA is detected, hybridization and washing can be performed with high stringency. , Such an operation is unnecessary.
Therefore, the necessity of these treatments was examined in the ISH of the tissue transferred to the nitrocellulose membrane.

According to the above method 1, frozen sections (5 μm thick) of hindlimb buds of 14-day-old fetal mice were transferred to a nitrocellulose membrane (BA85-PH75 nitrocellulose membrane) having a pore size of 0.05 μm and labeled with DIG rat II. It was hybridized with a collagen RNA probe (0.1 μg / ml). However, as the buffer, PBT buffer [0.1M sodium phosphate buffer containing 0.1% Tween 20 (NaPB), pH 7.2] was used, and alkaline phosphatase-conjugated DIG antibody and 4- Nitro blue tetrazolium chloride (NBT) and 5
-Bromo-4-chloro-3-indolyl phosphate (BCI
The probe was detected using P). In the preparation of the above sample, A: HCl of step (6) in method 1
When no treatment was carried out, B: HCl treatment in step (6) in Method 1 and acetylation in Steps (7) to (9) were conducted, C: HCl in Step (6) in Method 1 When the treatment, the acetylation of steps (7) to (9) and the ribonuclease treatment of steps (16) to (19) were not performed, D: a sample by four methods in the case where all steps of method 1 were performed Was produced.

As a result, A was detected by color development for 1 hour.
A strong signal was observed in the cartilage tissue in all the groups ~ D. In particular, in C that was not treated with ribonuclease, a sufficient signal was obtained in 10 minutes. In addition, background was hardly observed in any of the groups. Therefore, it was found that the method of the present invention does not require the conventionally required HCl treatment, acetylation and ribonuclease treatment, and the time required for the ISH method can be shortened.

Test Example 3 Examination of Optimal Concentration of Probe in ISH Method Using Nitrocellulose Membrane Generally, in the ISH method, the concentration of the probe used for hybridization is one of the important factors that influence the success of the detection method. Is. If the probe concentration is too low, the gene of interest may not be detected.
Too much will make the specific signal undetectable due to the high background signal. For example, a DIG-labeled RNA probe is usually used at a concentration of 0.1 to 1 μg / ml, but the optimum concentration varies depending on the individual probe and also varies depending on the hybridization conditions.
Therefore, type II collagen RNA in the method of the present invention
The optimum concentration of probe was investigated.

In the same manner as in Test Example 2C, except that the probe concentration was 0.3 μg / ml, 0.1 μg / ml, 30 ng / m 2.
Hybridization was performed at l, 10 ng / ml or 3 ng / ml. After washing the sample, it was reacted with an alkaline phosphatase-conjugated anti-DIG antibody to give NBT and BCIP.
And reacted for up to 9 hours. The background and signal strength at this time were evaluated by observation by two observers under a stereoscopic microscope. The sample was observed every 10 minutes and the time when a clear signal was obtained was measured. Table 2 shows the results
Shown in In Table 2, the background and signal strengths are evaluated as follows. Background ++++: Strong color development is observed throughout the tissue except cartilage. ++: Coloring is observed in the entire tissue except cartilage. +: Coloring is observed in some tissues other than cartilage. -: No coloring is recognized. Signal +++: Strong coloring is observed throughout the cartilage tissue. ++: Coloring is recognized throughout the cartilage tissue. +: Some cartilage tissue is colored. -: No coloring is recognized.

[0031]

[Table 2]

As shown in Table 2, 10 ng / ml to 0.1 μg
A specific signal could be observed in a wide range of / ml. However, at a concentration of 0.3 μg / ml or higher, the background increased due to the reaction for a long time, so that the optimum concentration of the type II collagen RNA probe in the method of the present invention is 3 ng / ml to 100 ng / ml. It is considered that this concentration is 1 / 10th of the optimum concentration of this probe in the conventional method.
It is 1/100.

Test Example 4 Comparison of Detection Sensitivity between the Conventional Method and the Method of the Present Invention Using the DIG-labeled RNA probes of rat aggrecan and type II collagen, the detection sensitivity of the conventional method and the method of the present invention were compared. Use a silane-coated glass slide or a nitrocellulose membrane with a pore size of 0.05 μm
Facial frozen sections (8 μm thick) or hindlimb bud frozen sections (8 μm thick) of day-old rat fetuses were placed, and the conventional method was carried out by the above method 3, and the method of the present invention was carried out by the above method 2. It was However, for the face-derived section, I
An aggrecan probe was used for a type I collagen probe and a hindlimb bud-derived section, and the buffer solution is as shown in Test Example 2. Then, color development was performed under the same conditions, and observation was performed every 5 minutes to measure the time when a clear signal was detected throughout the cartilage expressing type II collagen and aggrecan. The results are shown in Table 3.

[0034]

[Table 3]

As is clear from Table 3, in both cases of type II collagen probe and aggrecan probe, the method of the present invention confirmed the signal in a shorter time than the conventional method. In the case of the aggrecan probe, the signal obtained by the method of the present invention was stronger than that obtained by the conventional method even in the observation 8 hours after the color development. in this way,
It has been found that the method of the present invention is more sensitive than conventional methods and requires less probe.

Examination Using Various Probes The effectiveness of the method of the present invention was examined using DIG-labeled probes for rat type I collagen, type II collagen, type IX collagen and type X collagen. The hindlimb of a 14-day-old fetal mouse was used as a sample, and all probes were 100 ng.
ISH was performed according to Method 2 above using a concentration of / ml. As a result, for type I collagen, a strong signal was observed in the entire tissue except the epiphyseal cartilage, particularly in the bone tissue,
For type II collagen, a signal was observed in almost all regions of the cartilage tissue, for IX collagen, a specific signal was observed in the entire epiphyseal cartilage, and for type X collagen, it was close to bone among cartilage. A strong signal was observed in the so-called hypertrophic chondrocyte layer. The fact that specific signals were obtained with various probes in this way indicates that the method of the present invention is effective.

Example 1 Transfer of Biological Sample to Nitrocellulose Membrane A nitrocellulose membrane having a pore size of 0.05 μm was cut to an appropriate size, and then distilled water at 90 ° C. or 0.1 M CaCl _{2 was} added.
After soaking in the solution for 10 minutes, it was air dried. At the time of use, it was fixed to a slide glass with a clip. As a biological sample, for example, the limbs of a 4-week-old rat were embedded in OCT compound, and a frozen section with a thickness of 8 μm was prepared. Transfer was completed by placing the prepared frozen section on a nitrocellulose membrane and completely drying it with a dryer. Then 1
The cells were treated with 0 μg / ml proteinase K, refixed with 4% paraformaldehyde in PBT buffer at 4 ° C. for 20 minutes, and then used for hybridization. Type II collagen ISH The nitrocellulose membrane to which the prepared biological sample was transferred was cut into an appropriate size, and then placed in a tube or a multi-well plate that could be sealed. Hybridization buffer (50% formamide, 10 mM Tris-
HCl, pH 7.6, 1 mM EDTA, 0.2% SDS,
1x Denhardt's solution, 0.6M NaCl, 10% dextran sulfate, 200 µg / ml yeast tRNA) was added and prehybridization was performed at 65 ° C for 3 hours or more, and then digoxigenin-labeled type II collagen R was added.
NA probe was added, and hybridization was carried out at 65 ° C. overnight. After hybridization, 2 × SS
Washing was performed at 60 to 65 ° C with C and 50% formamide. After washing twice for 20 minutes, 0.2x
It wash | cleaned twice for 15 minutes at 60-65 degreeC using SSC. After that, an anti-digoxigenin antibody conjugated with alkaline phosphatase was reacted, and color was developed using NBT and BCIP for detection. In this experiment, a probe amount of 1/50 of the conventional method was used, and a signal with a low background and a sufficient intensity could be obtained with a detection time of 10 minutes. In addition, the background did not become high even when the color development reaction was continued for 6 hours.

[0038]

As described above, according to the present invention,
It is possible to provide an improved method for detecting a nucleic acid that solves the problems of the conventional ISH method, is simple, and has high sensitivity and has a wide range of applications.

Claims (9)

[Claims] 1. A method for detecting a nucleic acid, which comprises preparing a sample by detecting the presence of a target sequence in a nucleic acid in a sample by transferring and holding the sample on a thin film support. 2. The method of claim 1, wherein the thin film support is a polymeric film. 3. The method according to claim 2, wherein the thin film support is a nitrocellulose membrane. 4. The method according to claim 2, wherein the thin film support is a polymer film equivalent to a nitrocellulose film. 5. The method according to claim 1, wherein the transfer to the thin film support is performed by a freeze transfer method. 6. The method according to claim 1, wherein the detection of the presence of the target sequence is carried out by hybridization. 7. The method according to claim 1, wherein the presence of the target sequence is detected by a nucleic acid amplification method. 8. The method according to claim 7, wherein the presence of the target sequence is detected by the PCR method. 9. A thin film support as a member.
A kit for detecting nucleic acid for the method described in 1.