GENE EXPRESSION ANALYSIS METHOD USING TWO DIMENSIONAL cDNA LIBRARY

Patent metadata

Field	Value
Publication number	US-2017283796-A1
Application number	US-201715621316-A
Country	US
Kind code	A1
Filing date	Jun 13, 2017
Priority date	Dec 4, 2009
Publication date	Oct 5, 2017
Grant date	—

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

1 - 28 . (canceled)

29 . An analysis apparatus, comprising:
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 the support; a solution supplying unit; and an analyzing unit for conducting an analysis of a test nucleic acid, wherein the test nucleic acid-capturing part comprises a nucleic acid probe immobilized thereto, and the nucleic acid probe is positioned below a cell on the support; wherein the test nucleic acid extracted from the cell on the support by a nucleic acid extraction reagent, which is supplied by the solution supplying unit, is captured to the nucleic acid probe; and wherein a complementary strand of the captured test nucleic acid is synthesized on the support by an enzyme for the complementary strand synthesis, which is supplied by the solution supplying unit, such that a cDNA library is prepared on the support.

30 . The apparatus of claim 29 , wherein the analyzing unit obtains positional information of the test nucleic acid on the support, which has been captured to the nucleic acid-capturing part of the support.

31 . The apparatus of claim 30 , wherein the analyzing unit analyzes gene expression profile for the cell based on the positional information of the test nucleic acid.

32 . The apparatus of claim 31 , wherein the analyzing unit analyzes gene expression profile for each of a plurality of the cells, and compares the gene expression profiles of the cells.

33 . The apparatus of claim 29 , wherein the analyzing unit optically analyzes positional information of the test nucleic acid of the support, which has been captured to the nucleic acid-capturing part.

34 . The apparatus of claim 30 , wherein the analyzing unit analyzes the correlation between a cell shape and gene expression based on a microscopic image of the cell and the positional information of the test nucleic acid.

35 . The apparatus of claim 29 , wherein the support comprises cDNA library prepared by reverse transcription of the captured test nucleic acid.

36 . The apparatus of claim 29 , wherein the test nucleic acid is messenger RNA (mRNA).

37 . The apparatus of claim 36 , 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.

38 . The apparatus of claim 29 , 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 beads.

39 . The apparatus of claim 29 , 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.

40 . A device, comprising:
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 the support; and a solution supplying unit, wherein the test nucleic acid-capturing part comprises a nucleic acid probe immobilized thereto, and the nucleic acid probe is positioned below a cell on the support; wherein the test nucleic acid extracted from the cell on the support by a nucleic acid extraction reagent, which is supplied by the solution supplying unit, is captured to the nucleic acid probe; and wherein a complementary strand of the captured test nucleic acid is synthesized on the support by an enzyme for the complementary strand synthesis, which is supplied by the solution supplying unit, such that a cDNA library is prepared on the support.

41 . The device of claim 40 , wherein the support comprises cDNA library prepared by reverse transcription of the captured test nucleic acid.

42 . The device of claim 40 , wherein the test nucleic acid is messenger RNA (mRNA).

43 . The device of claim 42 , 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.

44 . The device of claim 40 , 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 beads.

45 . The device of claim 40 , 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.

Assignees

• Hitachi Ltd

Inventors

• Shirai Masataka
• Kambara Hideki
• Taniguchi Kiyomi

Classifications

• C12Q1/6841

  In situ hybridisation · CPC title

• G01N33/54306

  Solid-phase reaction mechanisms · CPC title

• C40B40/08

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

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C40B50/06

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title