GENE EXPRESSION ANALYSIS METHOD USING TWO DIMENSIONAL cDNA LIBRARY

a gene expression and two-dimensional technology, applied in the field of gene expression analysis methods, can solve the problems that the expression levels of a number of genes cannot be observed, and the satisfactory method has not yet been developed, and achieve the effect of simple and efficient detection

Inactive Publication Date: 2012-09-27
HITACHI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0045]The present invention provides a method for analyzing a gene expression profile. The method of the present invention enables to analyze expression of a gene in a sample with its two-dimensional positional information. Furthermore, in the method of the present invention, since genes expressed in a sample can be all converted into cDNAs to construct a cDNA library, gene expression can be simply and efficiently detected. Therefore, the present invention is useful for the fields of e.g., cell function analysis, biological tissue analysis, diagnosis of diseases and drug development. Particularly, by applying the present invention to pathological pieces so far collected, elucidation of various diseases, establishment of diagnostic approach, or development of pharmaceutical products, etc. can be performed.

Problems solved by technology

However, a satisfactory method has not yet been developed up to present.
If the number of target genes is one or two, gene expression in the tissue can be observed by use of a label using fluorescent probes; however, the expression levels of a number of genes cannot be observed.

Method used

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  • GENE EXPRESSION ANALYSIS METHOD USING TWO DIMENSIONAL cDNA LIBRARY
  • GENE EXPRESSION ANALYSIS METHOD USING TWO DIMENSIONAL cDNA LIBRARY
  • GENE EXPRESSION ANALYSIS METHOD USING TWO DIMENSIONAL cDNA LIBRARY

Examples

Experimental program
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example 1

[0113]This Example is an example of using a two-dimensional cDNA library sheet, which was constructed in a pore array sheet, from sheet-type cells while keeping positional information of mRNA contained therein. Hereinafter, a sheet in which a number of pores are two-dimensionally formed will be referred to as a pore array sheet and a pore array sheet in which a cDNA library is formed will be referred to as a cDNA library sheet. A conceptual figure of an example of the method is shown in FIG. 1.

[0114]This method contains: a step of extracting and trapping mRNA, while keeping cell positional information, by a DNA probe within a sheet (FIG. 1 (a)); a step of preparing a cDNA library by reverse transcription within the sheet (FIG. 1 (b)); a step of determining presence of a target cDNA by hybridizing a DNA probe (padlock probe), which is capable of hybridizing with the target cDNA at both ends, with the target cDNA and preparing a ring-form DNA probe (referred to as a ring probe) throug...

example 2

[0147]This Example is outlined in FIG. 11. This is an example of using a two-dimensional cDNA library sheet, which was constructed from a tissue slice sample 1101 in a pore array sheet. A frozen tissue slice was cut by a microtome into a film-form sample having a thickness of about 5 to 20 μm. As shown in FIG. 11, the tissue slice sample 1101 is mounted on a pore array sheet 1 made of alumina and immediately covered with a low melting-point agarose containing a cell lysis reagent. The agarose solution remains in a state of solution at 35° C. The sheet 1 is immediately gelatinized by cooling it to 4° C. Thereafter, the sheet is set in the reaction cell shown in FIG. 2, and mRNA is extracted in the same manner as Example 1.

[0148]A solution, which is prepared by mixing a low melting-point agarose gel (SeaPrep Agarose; gelatinization temperature is 19° C., melting point is 45° C. at an agarose concentration of 2%) and a cell lysis reagent, is used to lyse cell membrane and cell tissue w...

example 3

[0171]In this Example, unlike Example 1 or 2 in which a sheet having pores was used as a cDNA library sheet, another support such as a membrane was used to prepare a cDNA library.

[0172]As the membrane, a membrane rarely adsorbing a protein such as a cellulose acetate membrane, a nitrocellulose membrane or a membrane formed of a mixture of these and a nylon membrane can be available. In the Example, a case where a cellulose acetate membrane (Whatman) having a thickness of 115 μm, a pore size of 0.2 μm and a diameter of 25 mm will be described.

[0173]As shown in FIG. 18, to the surface of a membrane fiber 1802 within a membrane 1801, poly-T probes 1803 are immobilized by the silane coupling treatment in the same manner as in Example 1. At the same time, the MPC treatment is performed in the same manner. Next, the membrane is set in the same reaction cell as in Example 1 or 2. A tissue slice sample 1101 is mounted and the same treatments are performed. As a result, mRNA can be trapped b...

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Abstract

The present invention provides a method and/or means for collecting and analyzing an individual cell in a tissue, and at the same time, quantitatively monitoring the expression levels of various genes while keeping two-dimensional information in the tissue. Specifically, the present invention provides a method comprising preparing a cDNA library from mRNA while keeping two-dimensional cellular distribution information and obtaining the gene expression levels at any site or all sites at a level of single cell. More specifically, the present invention provides a method comprising preparing a cDNA library in a sheet-form from mRNA while keeping two-dimensional cellular distribution information and repeatedly using the cDNA library in the detection of the gene expression, thereby allowing measurement of the expression distribution for a number of genes at a high accuracy.

Description

BACKGROUND OF THE INVENTION[0001]1. Technical Field[0002]The present invention relates to a gene expression analysis method. More specifically, the present invention relates to a method of preparing a cDNA library from a nucleic acid contained in a sample and analyzing a gene expression profile in the sample.[0003]2. Background Art[0004]In gene expression analysis, mRNA is collected from cells and a complementary strand thereof, i.e., cDNA is prepared. This is amplified by a polymerase chain reaction (PCR) to increase the number of copies. Thereafter, a target is trapped by the corresponding site of a probe by use of a DNA probe array (DNA chip) and detected based on fluorescence. However, in such a method using PCR amplification and a DNA chip, accuracy of quantitative analysis is low, and thus, a highly accurate analysis method for a gene expression profile has been desired. With completion of human genome analysis, a need for quantitative analysis of gene expression has been incr...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B30/04
CPCC12N15/1093C12Q1/6809C12Q1/6837C40B40/08C40B50/06G01N33/54306G01N2458/10C12Q2531/125C12Q2533/107C12Q2565/537C12Q1/6841C12Q2565/514
Inventor SHIRAI, MASATAKAKAMBARA, HIDEKITANIGUCHI, KIYOMI
Owner HITACHI LTD
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