Generation of Multiple mRNA Fingerprints Using Fluorescence-Based Differential Display and an Automated DNA Sequencer

Abstract

Differential display is a method for the survey, analysis and comparison of gene expression in eukaryotic cells and tissues. Differential display involves isolation of high-quality nondegraded RNA, selective reverse transcription of polyadenylated mRNA using specific anchored oligopolydeoxythymidine [oligo(dT)] primers, and the subsequent PCR amplification of the cDNA with the same oligo(dT), an arbitrary upstream primer and radioisotopes for labeling the PCR products. The radioisotopically labeled products are then separated on a sequencing gel. In this report, we describe a rapid, specific, nonradioactive fluorescent differential display methodology in which fluorescently differentially labeled anchored oligo(dT) downstream primers are used in the reaction, with subsequent analysis of fluorescently labeled PCR products on an automated sequencer. Complete gene expression profiles, containing multiple mRNA fingerprints are possible by the simultaneous comparison of the multicolored banding patterns of the fluorescently differentially labeled products from several primer combinations. This modification of the differential display technique simplifies the assay and increases the throughput of high sample volumes required for comparative gene expression studies in various clinical applications.