18] Identification of differentially expressed genes using RNA fingerprinting by arbitrarily primed polymerase chain reaction

Abstract

Genomic fingerprinting by arbitrarily primed polymerase chain reaction (AP-PCR) is a sensitive and efficient method for generating a large number of molecular markers. Based on the selective amplification of DNA sequences flanked, by chance, by sequences matching an arbitrarily chosen primer, AP-PCR reveals sequence polymorphisms between different template DNAs. The ability to detect differences between fingerprints of closely related organisms made this approach a useful tool for studying genetic diversity, population biology, epidemiology, and genetic mapping. The more recent application of AP-PCR fingerprinting to RNA, differential display, or RNA-arbitrarily primed PCR (RAP-PCR) has resulted in an interesting tool for the detection of differential gene expression. This chapter focuses on the use of RAP-PCR fingerprinting. It describes the method and provides the present protocols for the different steps from initial RNA preparation through sequence analysis and confirmation of differential expression of the transcripts identified.