A Protocol for Nonradioactive Differential Display Tested on Carrot Auxin-Resistant Mutants

Abstract

A new protocol for nonradioactive differential display visualized by ethidium bromide staining was developed for carrot total RNA. The procedure was used to identify genes specifically expressed in carrot auxin-resistant mutants compared to their wild-type. Different populations of total RNAs were reversely transcribed using three anchored oligo-dT primers. PCR amplification of relative cDNAs was carried out in combination with five arbitrary primers. New polyacrylamide gel properties (lower matrix concentration, higher thickness, nondenaturing gel and ethidium bromide staining) were combined to provide a simplified protocol that can resolve amplified transcripts over a large range of molecular weights. Several specific transcripts were successfully identified by this procedure.