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Gene expression analysis using array with immobilized tags of more than 25 bp (SuperSAGE-Array)

Inactive Publication Date: 2007-07-26
IWATE PREFECTURAL GOVERNMENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006] It is an object of the present invention to provide a method of gene expression analysis that enables extensive gene expression analysis and simultaneous analysis of multiple samples of organisms for which the genomic analysis has not yet been advanced.
[0007] In order to attain the above object, the present inventors had examined whether or not tags each of 26 bp of SuperSAGE (SuperSAGE tags) could be utilized as probes for microarrays. As a result, they discovered that the results of gene expression analysis attained with the use of an array with immobilized SuperSAGE tags would be similar to those attained via conventional SAGE™, and that such results could be attained through a single hybridization step. Further, they also discovered that immobilization of SuperSAGE tags would produce unexpected effects, i.e., preparation of microarrays would be remarkably facilitated in non-model organisms for which no EST, cDNA, or genomic sequences are available.
[0021] The present invention facilitates the extensive gene expression analysis and simultaneous analysis of multiple samples of organisms for which genomic analysis has not yet been advanced. Accordingly, the present invention overcomes the drawbacks of conventional microarray or SAGE™ systems and realizes extensive analysis of known and unknown genes.

Problems solved by technology

With the use of a microarray, however, expression analysis can only be conducted exclusively for the genes spotted on the array.
This requires large amounts of time and cost.
However, SAGE™ is not substantially suitable for simultaneous analysis of multiple samples due to the large number of experimental steps required.
Thus, applications of such tags are restricted to model organisms.

Method used

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  • Gene expression analysis using array with immobilized tags of more than 25 bp (SuperSAGE-Array)
  • Gene expression analysis using array with immobilized tags of more than 25 bp (SuperSAGE-Array)
  • Gene expression analysis using array with immobilized tags of more than 25 bp (SuperSAGE-Array)

Examples

Experimental program
Comparison scheme
Effect test

example 1

1. Material and Method

1) Preparation of RNA

[0055] For rice SuperSAGE and oligoarray systems, rice leaves (variety: Yashiromochi) and suspension-cultured cells (variety: Kakehashi) were prepared. For the oligoarray system, mRNA was extracted from the rice (variety: Yashiromochi) and the cultured cells (variety: Kakehashi) 1 month after sowing using an mRNA Purification Kit (Amersham Pharmacia).

[0056] For Nicotiana benthamiana SuperSAGE and oligoarray systems, leaves into which Agrobacterium containing the following plasmids had been injected were prepared. Two days after the Agrobacterium injection, Nicotiana benthamiana leaves were treated with dexamethasone (DEX), and mRNA was extracted using the mRNA Purification Kit (Amersham Pharmacia) 4 hours later.

2) Plasmids

[0057] NbCD1 (JP Patent Publication (Unexamined) No. 2005-278634) and NbCD3 cDNA (JP Patent Publication (Unexamined) No. 2005-245251), which had been isolated by the present inventors in the past, were used. A bina...

example 2

[0093] In Example 1, model rice plants were subjected to expression analysis using an array with 41 SuperSAGE tags immobilized thereon, and non-model Nicotiana benthamiana plants were subjected to expression analysis using an array with 154 SuperSAGE tags immobilized thereon. In both cases, the results of analysis were very consistent with the results of expression analysis via SuperSAGE. In this example, an array with 1,000 SuperSAGE tags immobilized thereon was prepared for rice, and the results of expression analysis via SuperSAGE-Array were compared with those via SuperSAGE.

[0094] In accordance with the procedure of Example 1, mRNAs were extracted from rice leaves (variety: Yashiromochi) and cultured cells of rice (variety: Kakehashi) to prepare SuperSAGE libraries. From these libraries, 1,000 SuperSAGE tag sequences were selected. Among them, 78 tags represented equally expressed genes, 438 tags were more prevalent in leaves and 484 tags were more abundant in suspension-cultur...

example 3

[0098] Among the tags that were found to be expressed at high levels in all of the NbCD1- and NbCD3-overexpressing Nicotiana benthamiana leaves by the SuperSAGE-Array-based expression analysis in Example 1, 5 tags showing no sequence matches to known cDNA or EST were selected (NbCD3U14, 20, 25, 32, and 40), and identification of the genes corresponding thereto was attempted.

[0099] Full-length sequences of the tags were determined by the 3′-RACE and 5′-RACE methods in the following manner. As a template, RNA was isolated from NbCD3-overexpressing Nicotiana benthamiana leaves, and the RNA was flanked by adaptor sequences to synthesize cDNA. Based on the SuperSAGE tag sequences, a gene specific PCR primer and a primer complementary to the adaptor sequence were used to amplify a partial cDNA fragment from template cDNA. A primer was prepared based on the 5′-sequence of the resulting fragment, and the resulting primer and the adaptor primer (i.e., a primer complementary to the adaptor s...

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Abstract

This invention provides a method of gene expression analysis that enables extensive gene expression analysis and simultaneous analysis of multiple samples of organisms for which genomic analysis has not yet been advanced. In this method, tags each comprising an oligonucleotide of more than 25 bp for identifying expressed genes, wherein the 3′-end of the tag is defined by a cleavage site of a type III restriction enzyme and the 5′-end thereof is defined by a cleavage site of another restriction enzyme located closest to the 3′-end of the cDNA of such genes, are immobilized on a solid support, gene-containing samples are hybridized to the solid support, and the signals emitted from the genes hybridized to the tags are detected to analyze the gene expression profiles in the samples.

Description

CLAIM PRIORITY [0001] The present application claims priority from Japanese applications JP2005-359366 filed on Dec. 13, 2005 and JP2006-138515 filed on May 18, 2006, the content of which is hereby incorporated by reference into this application. TECHNICAL FIELD [0002] The present invention relates to a method of gene expression analysis. More particularly, the present invention relates to a method of gene expression analysis that enables highly reproducible and high-throughput analysis, with the use of a microarray with immobilized improved SAGE™ tags of more than 25 bp. BACKGROUND ART [0003] Techniques for transcript analysis, such as microarray analysis and serial analysis of gene expression (SAGE™), are indispensable for various types of biological research. Use of a microarray enables the expression analysis of large quantities of genes at one time and simultaneous analysis of multiple samples. With the use of a microarray, however, expression analysis can only be conducted exc...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12M1/34
CPCC12Q1/6837
Inventor MATSUMURA, HIDEOTERAUCHI, RYOHEI
Owner IWATE PREFECTURAL GOVERNMENT
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