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A strategy to identify differentially expressed genes using representational difference analysis and cDNA arrays.

S Kim¹, K Zeller, C V Dang

¹Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205, USA.

Analytical Biochemistry

|January 12, 2001

Summary

This study enhances gene discovery in c-Myc transgenic mouse liver by combining Representational Difference Analysis (RDA) with cDNA arrays. The improved method identifies both abundant and rare differentially expressed genes, including a novel one.

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Area of Science:

Molecular Biology
Genomics
Gene Expression Analysis

Background:

Identifying differentially expressed genes is crucial for understanding biological processes.
Representational Difference Analysis (RDA) coupled with cDNA arrays is a known method for gene discovery.
Optimizing probe strategies for cDNA arrays can improve the detection of low-abundance transcripts.

Purpose of the Study:

To identify differentially expressed genes in c-Myc transgenic mouse liver.
To compare the efficacy of radiolabeled cDNA pools versus radiolabeled difference products (DP2) from RDA for probing cDNA arrays.
To develop a more comprehensive method for detecting both abundant and rare differentially expressed transcripts.

Main Methods:

Representational Difference Analysis (RDA) was performed on c-Myc transgenic and normal mouse liver.
Difference products (DP2) and cDNA pools were generated and radiolabeled.
Commercial and custom cDNA arrays were probed using both radiolabeled cDNA pools and DP2 products.

Main Results:

Probing with DP2 products identified low-abundance transcripts missed by cDNA pools.
DP2 probes detected highly differentially expressed abundant transcripts but missed some abundant transcripts with low differential expression.
Combining radiolabeled cDNA and DP2 products provided a comprehensive identification of differentially expressed transcripts (abundant and rare) and reduced false positives.

Conclusions:

A combined approach using radiolabeled cDNA and DP2 probes significantly enhances the comprehensive identification of differentially expressed genes.
This method is effective for discovering both abundant and rare transcripts, including novel genes.
The study successfully identified 16 differentially expressed genes in c-Myc transgenic mouse liver, with one novel gene identified.