Gene expression analysis ME1HOD using two dimensional cDNA library

Patent metadata

Field	Value
Publication number	US-11214796-B2
Application number	US-201715621316-A
Country	US
Kind code	B2
Filing date	Jun 13, 2017
Priority date	Dec 4, 2009
Publication date	Jan 4, 2022
Grant date	Jan 4, 2022

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.

First claim

What is claimed is:

1. An analysis apparatus, comprising:
a test nucleic acid and a nucleic acid extraction reagent;
a support where a test nucleic acid-capturing part has been two-dimensionally distributed, wherein the test nucleic acid-capturing part is on the surface of and/or within a plurality of pores perpendicular to the support;
a nucleic acid probe of the test nucleic acid-capturing part;
a captured test nucleic acid having a complementary strand;
an enzyme for synthesizing the complementary strand;
a channel for feeding at least test nucleic acids and reagents into the apparatus;
an optical system for conducting an analysis of the test nucleic acid;
a cell on the plurality of pores perpendicular to the support; and
a cDNA library,
wherein the test nucleic acid-capturing part comprises the nucleic acid probe immobilized thereto, and the nucleic acid probe being positioned below the cell on the support;
wherein the nucleic acid probe is capable of capturing the test nucleic acid that is considered captured by virtue of its interaction with the nucleic acid probe and the test nucleic acid on the plurality of pores perpendicular to the support; and
wherein the cDNA library on the support is a product of synthesis of the complementary strand of the captured test nucleic acid by the enzyme for synthesizing the complementary strand.

2. The apparatus of claim 1 , wherein the optical system is configured to obtain lateral positional information of the test nucleic acid on the support, which has been captured to the nucleic acid-capturing part of the support.

3. The apparatus of claim 2 , wherein the optical system is configured to analyze gene expression profile for the cell based on the lateral positional information of the test nucleic acid.

4. The apparatus of claim 3 , wherein the optical system is configured to analyze gene expression profile for each of a plurality of the cells, and compares the gene expression profiles of the cells.

5. The apparatus of claim 1 , wherein the optical system is configured to optically analyze lateral positional information of the test nucleic acid of the support, which has been captured to the nucleic acid-capturing part.

6. The apparatus of claim 2 , wherein the optical system is configured to analyze the correlation between a cell shape and gene expression based on a microscopic image of the cell and the lateral positional information of the test nucleic acid.

7. The apparatus of claim 1 , wherein the test nucleic acid is messenger RNA (mRNA).

8. The apparatus of claim 7 , wherein the test nucleic acid-capturing part of the support comprises a DNA probe containing a poly-T sequence immobilized thereto as the nucleic acid probe.

9. The apparatus of claim 1 , wherein the support is at least one selected from the group consisting of a sheet, a membrane, a gel thin film, a capillary plate and packed beads.

10. The apparatus of claim 1 , wherein the support is a porous sheet, and the test nucleic acid-capturing part is a pore of 20 nm to 200 nm in diameter, and wherein the nucleic acid probe is immobilized to the pore which is perpendicular to the support.

11. A device, comprising:
a test nucleic acid and a nucleic acid extraction reagent;
a support where a test nucleic acid-capturing part has been two-dimensionally distributed, wherein the test nucleic acid-capturing part is on the surface of and/or within a plurality of pores perpendicular to the support;
a nucleic acid probe of the test nucleic acid-capturing part;
a captured test nucleic acid having a complementary strand;
an enzyme for synthesizing the complementary strand;
a channel for feeding at least test nucleic acids and reagents into the apparatus;
a cell on the support; and
a cDNA library;
wherein the test nucleic acid-capturing part comprises the nucleic acid probe immobilized thereto, and the nucleic acid probe is positioned below the cell on the support;
wherein the nucleic acid probe is configured to capture the captured test nucleic acid that is considered captured by virtue of an interaction with the nucleic acid probe and the cDNA library on the plurality of pores perpendicular to the support; and
wherein the cDNA library on the pores perpendicular to the support is a product of synthesis of the complementary strand of the captured test nucleic acid by the enzyme for synthesizing the complementary strand.

12. The device of claim 11 , wherein the test nucleic acid is messenger RNA (mRNA).

13. The device of claim 12 , wherein the test nucleic acid-capturing part of the support comprises a DNA probe containing a poly-T sequence immobilized thereto as the nucleic acid probe.

14. The device of claim 11 , wherein the support is at least one selected from the group consisting of a sheet, a membrane, a gel thin film, a capillary plate and packed beads.

15. The device of claim 11 , wherein the support is a porous sheet, and the test nucleic acid-capturing part is a pore of 20 nm to 200 nm in diameter, and wherein the nucleic acid probe is immobilized to the pore which is perpendicular to the support.

Assignees

• Hitachi Ltd

Inventors

• Shirai Masataka
• Kambara Hideki
• Taniguchi Kiyomi

Classifications

• C12N15/1093 Primary

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• G01N2458/10

  Oligonucleotides as tagging agents for labelling antibodies · CPC title

• G01N33/54306

  Solid-phase reaction mechanisms · CPC title

• C12Q1/6809

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• C40B40/08

  Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries · CPC title