JP2001299394A - Comparative detection of amounts of rna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of comparatively determining the amount of the RNA of which the expression frequency can be measured between multiple tissues by simple operation, reusing the same samples and obtaining a large amount of data with small amounts of sample specimens and can measure the gene fragments other than the 3'-terminus through the ATAC technique. SOLUTION: A single strand nucleotide corresponding to the adaptor sequence according to the ATAC technique is fixed on the plate that can be used in the PCR, and the asymmetric PCR including the sample for the ATAC technique that is finished the process until the adaptor ligation is carried out to extend the nucleotide, whereby the operations are simplified, the reuse of the same samples becomes possible and a large amount of data can be obtained from a small amount of samples and the gene fragments other than 3'-terminus can be measured according to the ATAC technique. The sample according to the ATAC technique in which the originating tissue, the restriction enzyme on the cleavage of the cDNA are fixed to the multi-perforated plate whereby the expression frequency between multiple tissues can be measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for comparatively detecting RNA, and more particularly to a method for comparatively detecting RNA performed by fixing an adapter-added cDNA to a PCR reactor.

[0002]

2. Description of the Related Art One of the methods for measuring the expression frequency of a gene is Adapter Tagged Competitive PCR (Kato, K .;
(1997) Nucleic Acid Res. , 25,4694- 4696.
Hereinafter, referred to as ATAC method). This ATA
Method C uses the polymerase chain reaction (Polymerase chain r)
eaction (hereinafter referred to as PCR), which is more sensitive than Northern hybridization, which is generally a technique for measuring the level of gene expression.

[0003] The ATAC method is known as a highly quantitative technique by preparing and mixing adapter-added cDNA from RNA for preparing a standard curve and RNA to be detected, the concentration of which is adjusted stepwise. In this method, a standard curve is prepared by adding adapters having different lengths to cDNAs derived from the same tissue and changing the concentration stepwise during mixing.

[0004] Fig. 3 shows the size of a fluorescently labeled PCR product separated by gel electrophoresis.
This is an example in which the amount of the R product is detected. In the ATAC method using six types of adapters, the first adapter is set to 1 time the standard tissue, the third adapter is set to 3 times the same tissue, A standard curve is prepared by setting the sixth adapter to a concentration 10 times the concentration. A cDNA fragment derived from the tissue to be measured is added to the second, fourth, and fifth adapters, and the expression ratio of RNA to the standard tissue is detected from the prepared standard curve.

[0005]

However, the above A
In the TAC method, operations such as ligation of an adapter and mixing of an adapter-added cDNA (hereinafter, referred to as a sample) are complicated, and c-derived from a tissue used for measurement.
When DNA is disposable and there is only a small amount of tissue-derived RNA, the number of measurements is limited, and a sample is prepared for each measurement and used as a template for PCR.
The number of tissues whose expression frequency can be measured according to the number of available adapters is limited because it may be difficult to compare measured values or confirm experiments using the same sample, and to distinguish tissues based on adapter length. Since the 3 ′ end cDNA fragment is used for measurement, if the 3 ′ end sequence is unknown, gene-specific primers cannot be designed and ATAC
There were difficulties such as the inability to use the law.

[0006] Further, there is a drawback in that the number of different adapters that can be used for detection or the preparation of samples is complicated, and the number of tissues that can measure the expression ratio of a large amount of genes effectively is limited. Was.

[0007] The present invention solves the above-mentioned drawbacks of the conventional method for measuring the expression frequency of a gene.
It is an object of the present invention to provide a method for comparatively detecting the amount of RNA capable of measuring the gene fragment other than the terminal by the ATAC method and measuring the expression frequency between multiple tissues.

[0008]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the method for comparatively detecting the amount of RNA according to the present invention is a method for preparing a single tube corresponding to the adapter sequence of the ATAC method in a tube usable for PCR. An asymmetric PC containing a sample of the ATAC method in which the nucleotides of the strand are fixed and the process up to the ligation of the adapter is completed.
By performing R, the extension of nucleotides enables simplification of experimental operations, reuse of the same sample, and acquisition of large amounts of data with small samples.
Furthermore, it has been found that the fixation of a gene fragment other than the 3 'end enables measurement by the ATAC method. In addition, it is possible to measure the expression frequency between multiple tissues by fixing the sample of the ATAC method in which the source tissue and the restriction enzyme at the time of cDNA cleavage are changed to a multi-well plate such as a 96-well plate by the above method. Is found.

That is, the present invention comprises the following steps:
A different type of adapter is added to each of a plurality of cDNAs obtained by reverse transcription from the same or different tissue RNA, and the adapter-added cDN obtained by (b) and (a) is added.
A. Mixing A and adding cD to the reactor for PCR
DNA derived from NA or adapter-added cDNA
Immobilizing the fragment, (c) amplifying using an adapter primer having a part of the sequence of the adapter and a gene-specific primer, and (d) measuring the quantitative ratio of cDNA between multiple tissues,
(E) washing the reactor after amplification of the adapter-added cDNA or the DNA fragment derived from the adapter-added cDNA, which is a method for comparatively detecting the amount of RNA to detect RNA from the measurement results of (d), and Alternatively, a comparative detection method of the amount of RNA to be amplified using different adapter primers and gene-specific primers is also included.

[0010] Also, cDNA with an adapter added to the reactor
Alternatively, in order to fix a DNA fragment derived from an adapter-added cDNA, a method is used in which an oligonucleotide containing an adapter sequence is immobilized in a reactor, and the immobilized oligonucleotide is extended using a separately mixed adapter-added cDNA. Is adopted.

[0011] As different types of adapters, nucleotides having different lengths from each other are employed.

[0012] Different types of adapters are added, and adapter-added cDNAs prepared from cDNAs of the same tissue whose concentration is adjusted stepwise can be used.

Furthermore, a common adapter-added cDNA and a different adapter-added cDNA for each reactor can be used in two or more reactors.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a method for comparatively detecting the amount of RNA according to the present invention will be described in detail with reference to the drawings.

The method for comparatively detecting the amount of RNA according to the present invention comprises:
Adapto performs RNA detection by performing competitive PCR on cDNA to which different types of adapters have been added.
r Tagged Competitive PCR (ATAC method) simplifies the process by fixing the adapter-added cDNA to a PCR plate, and allows the adapter-added cD
This makes it possible to use NA repeatedly.

As shown in FIG. 1, the ATAC method using the method for comparatively detecting the amount of RNA according to the present invention uses cDNA from RNA containing a poly A sequence extracted from two tissues.
Samples A and B are synthesized. "-B" in the figure indicates that biotin is bound to the oligo dT primer used at the time of cDNA synthesis. Both the samples A and B are fragmented using the restriction enzyme Mbo-I, and the samples A and B each contain a common sequence and are ligated to adapters A and B having different lengths. Adapter-added cD containing 3'-terminal fragment
NA is selectively separated by binding to biotin using streptavidin-bound magnetic beads.

The cDNAs with the adapters obtained from Samples A and B are mixed so as to have the same concentration, and transferred to a tube as a reactor in which an oligonucleotide having a sequence common to the adapter is fixed on the inner wall in advance. An enzyme and a buffer necessary for PCR are added, and PCR is performed using the oligonucleotide fixed in the tube as a primer.

As a result, the adapter-added cDNA is fixed to the tube while maintaining the mixing ratio at the time of mixing.

When a new PCR is performed by adding an adapter primer containing a common sequence of the adapter and a gene-specific primer designed from the specific sequence of the gene to be detected, the abundance ratio of the specific gene in sample A and sample B A PCR product corresponding to the above is obtained. However, the restriction enzymes used, the number of samples, the fragment portion, and the selection method are not limited to the examples shown in the figures.

According to the method for comparatively detecting the amount of RNA of the present invention, by fixing a sample for preparing a standard curve and a different sample for each tube to many tubes, a large number of tissues can be obtained in a shorter time than the conventional method. It is possible to detect the amount of RNA during the period.

As shown in FIG. 2, a standard sample whose concentration is changed stepwise and three types of samples to be measured are fixed to each well of a 96-well plate as a reactor. By fixing each 96-well sample so as to contain samples derived from three different tissues, the expression frequency among 288 tissues can be measured. In this case, the same gene-specific primer is used for all of the 96 wells. Alternatively, A in FIG.
A sample of 36 tissues was fixed using 12 holes in a row, and B
Rows, C,..., H are the same as row A, and the expression of 8 genes can be detected among 36 tissues by using different gene-specific primers for each row. Moreover, since the sample fixed on the plate is not lost by washing the plate, it can be used for repeated measurement, and even in tissues and cells where only a small amount of RNA can be obtained, more genes can be used than in the conventional method. Can be measured.

Hereinafter, each step of the method for comparatively detecting the amount of RNA according to the present invention will be described. (1) Fixation of Adapter Sequence to Reactor An oligonucleotide having a specific sequence is fixed to a PCR-capable tube. Here, the specific sequence means a sequence matched with a part of the sequence of the adapter,
20 to 25 bases are preferred, but the length is not particularly limited.

The reactor may be a single tube or a well such as a 96-well plate (microplate).

The method for comparatively detecting the amount of RNA according to the present invention comprises:
Since it is possible to simultaneously measure the ratio of the amount of RNA between multiple tissues, it is preferable that oligonucleotides are evenly and equally fixed to the wells of a multi-well plate. The oligonucleotide to be immobilized can be obtained by a known method, for example, by chemical synthesis using a DNA synthesizer manufactured by PE Biosystems Japan. The sequence of the oligonucleotide to be immobilized may be the same or different in all of the tubes used for measurement. When different types of adapters do not have a common sequence, two or more types of oligonucleotides may be immobilized in one tube.

In fixing the oligonucleotide,
Known methods, that is, a method by physical adsorption, a method of directly cross-linking to the tube surface, a method by biotin-streptavidin or antigen-antibody reaction, and a method of directly synthesizing on the tube surface can be used. (2) Preparation of RNA and cDNA cDNAs corresponding to each RNA are synthesized from cells or tissues of various organs using reverse transcriptase. cD
The synthesis of NA can be performed by any known technique. For example, there are a method of preparing polyA + RNA and allowing it to act on reverse transcriptase, a method of preparing it using a commercially available kit (such as Lifetech Oriental), and the like.

At the time of cDNA synthesis from poly A + RNA, it is possible to select the 3 'end. For example, it is preferable to use an oligo dT primer with biotin and to select only the 3 'terminal side by magnet beads using binding with streptavidin, but this method is not limited. (3) Preparation of cDNA with Adapter The cDNA synthesized from each tissue is fragmented individually using the same restriction enzymes. From these, fragments derived from 5 to 7 types, preferably 6 types of tissues are selected, and
Different types of adapters having a sequence complementary to the oligonucleotide fixed in (1) and enabling ligation with cDNA fragmented with the restriction enzyme used are individually ligated. When adapters of different types have different lengths, the adapters having different lengths are designed to have a sequence of about 20 to 25 bases which are continuously common.

Six types of cDNAs with adapters are mixed at an appropriate ratio. Equal amounts of 6 different types of tissues are mixed, or adapters of different lengths are added to cDNAs from the same tissue, and the concentration is changed step by step to mix them as a standard tissue. Can be considered. Here, the type of organization is not limited to six.

When an oligo dT primer with biotin is used, it is possible to select only an adapter-added cDNA containing a 3 'terminal sequence. (4) Transfer of ATAC method sample to tube by extension reaction (1) Tube with (3) adapter-added cDNA
A mixture or a mixture obtained by extracting only the cDNA 3′-terminal fragment, an enzyme for PCR (such as Taq Polymerase),
dNTPs (dATP, dTTP, dGTP and dCT
P), a buffer for PCR is added, and PCR using only one side of the primers with the immobilized oligonucleotide as a primer is performed for several cycles. As a result, in the tube of (1), even with the same gene, a type such as length differs depending on the tissue of origin, and an adapter-added cDNA fragment reflecting the expression frequency of the gene group in each tissue is contained. It has been replaced by a fixed state. Here, the immobilized DNA can be made single-stranded by a denature operation. In the case of a mixture from which only the 3′-terminal fragment has been removed, the immobilized oligonucleotide can be extended while amplifying the sample by adding an appropriate amount of oligo dT primer during PCR. (5) Amplification An amplification reaction (PCR) is performed using the adapter primer and the gene-specific primer on the tube with the adapter-added cDNA fixed as prepared above. here,
An adapter primer is an oligonucleotide having a sequence common to an adapter. In the case of adapters having different lengths, these are oligonucleotides having a sequence complementary to a part of the common sequence which all the adapters have. Therefore, the adapter primer and the oligonucleotide for fixing to the tube may have a common length and sequence.

As for the conditions for PCR, a known ATAC
Method settings (eg, Kato, K. (1997) Nucleic Ac
id Res. , 25,4694-4696). (6) Detection The product obtained by amplification can be detected by a known method after transferring the amplification product in (5) to a fresh tube prepared separately. For example, by labeling the adapter primer used for PCR with a fluorescent dye in advance, the measurement can be performed by measuring the fluorescence intensity.

When adapters having different lengths are used, agarose gel electrophoresis, polyacryl gel electrophoresis, or the like is performed, and after staining, the intensity of the band of the amplified product can be measured. The amplification product can also be measured with a predetermined measuring device (a DNA analyzer manufactured by PE Biosystems, a sequencer manufactured by the company, etc.).

When the band intensity of the amplification product is measured as the fluorescence intensity, the ratio of the intensity indicates the amount of the cDNA with the adapter before amplification, that is, the ratio of the amount of RNA in each tissue. Can be compared and detected.

[0032]

According to the method for comparatively detecting the amount of RNA according to the present invention, complicated operations such as ligation of an adapter and mixing of cDNA with an adapter are not required.
Since the cDNA derived from the tissue used for measurement can be repeatedly used, the number of measurements can be increased even when the amount of tissue RNA is small, and it is not necessary to prepare a sample for each measurement and use it as a template for PCR. Tissue that allows comparison between values and confirmation experiments with the same sample, expression frequency can be measured according to the number of available adapters by fixing a common adapter-added cDNA to two or more tubes The limitation of the number is eliminated, and cDNA fragments other than the 3 'end can be fixed to the tube.
Even when the sequence at the 3 'end is unknown, gene-specific primers can be designed, and the ATAC method known as a highly quantitative technique can be effectively used.

[Brief description of the drawings]

FIG. 1 shows a procedure for immobilization of an adapter-added cDNA in an ATAC method using a method for comparatively detecting the amount of RNA according to the present invention.

FIG. 2 shows an ATA using a 96-well plate according to the present invention.
The method for fixing the adapter-added cDNA when detecting gene expression among multiple tissues by the method C is shown.

FIG. 3 shows the principle of measuring the gene expression frequency ratio using a standard tissue having a stepwise concentration in the ATAC method.

Claims (6)

[Claims] (1) different types of adapters are added to each of a plurality of cDNAs obtained by reverse transcription from the same or different tissue RNAs; and (b) the adapter-added cDN obtained by the above (a).
A and mixing the adapter-added cDNA or the adapter-added cD into a reactor for carrying out the polymerase chain reaction.
Immobilizing a DNA fragment derived from NA, (c) amplifying using an adapter primer having a part of the adapter sequence and a gene-specific primer, and (d) measuring the quantitative ratio of cDNA between multiple tissues. (E) a method for comparatively detecting the amount of RNA, wherein the method comprises detecting RNA from the measurement result of (d). 2. The reactor after the amplification of the adapter-added cDNA or the DNA fragment derived from the adapter-added cDNA is washed, and amplified using the same or different adapter primer and gene-specific primer as in (c). 2. The RNA according to claim 1, wherein
A comparative detection method of the amount. 3. The cDN with the adapter added to the reactor.
A or DN derived from the adapter-added cDNA
The method for immobilizing the fragment A comprises immobilizing the oligonucleotide containing the sequence of the adapter in the reactor, and extending the immobilized oligonucleotide using a separately added adapter-added cDNA. Item 7. A method for comparatively detecting the amount of RNA according to Item 1. 4. The method according to claim 1, wherein the different types of adapters are nucleotides having different lengths from each other. 5. A cDNA of the same tissue to which the different types of adapters are added, and the concentration of which is adjusted stepwise.
The method for comparatively detecting the amount of RNA according to claim 1, wherein an adapter-added cDNA prepared from the above is used. 6. The method according to claim 6, wherein said two or more reactors have a common adapter-added cDNA and a different adapter-added c for each reactor.
2. The RNA according to claim 1, wherein DNA is used.
A comparative detection method of the amount.