Differential Display Techniques to Identify Tumor Suppressor Gene Pathways

Differential display is a reverse-transcription polymerase chain reaction (RT-PCR) technique that was introduced in 1992 by Liang and Pardee (1 ). In this technique, messenger RNA 3′ termini are amplified using an anchored oligo-dT primer and a series of arbitrary 13-mers; a single round of cDNA synthesis is followed by PCR amplification in the presence of radiolabeled nucleotide, resulting in the amplification of subsets of mRNAs expressed in the cell. This complex PCR product is resolved on a denaturing polyacrylamide gel; following autoradiography, gene expression is detected as a ladder of cDNAs that differ in size by a single nucleotide. Theoretically, the use of different sets of primers from both directions in discrete RT-PCR reactions allows for the examination of the majority of expressed genes within the cell. This chapter will focus first on a brief review of the use of differential display to identify p53 target genes, as well as secondary tumor suppressor genes important for tumor progression. This will be followed by an overview of the methodology of differential display, along with recent advances in this technique; a more comprehensive analysis can be found in Methods in Molecular Biology, Volume 85: Differential Display Methods and Protocols , edited by Liang and Pardee (2 ). An outline of the methodology involved in differential display is provided in Fig. 1 , and individual steps are described below; typically these steps follow the protocol described by Liang and Pardee (1 ,2 ), but in some instances changes have been made and noted.