Yongjig Cho Susanne Stein Roger S Jackson II and Peng Liang

Summary

Differential display (DD) is one of the most commonly used approaches for identifying differentially expressed genes. Despite the great impact of the method on biomedical research, there has been a lack of automation of DD technology to increase its throughput and accuracy for a systematic gene expression analysis. Most of previous DD work has taken a "shotgun" approach of identifying one gene at a time, with limited polymerase chain reaction (PCR) reactions set up manually, giving DD a low-technology and low-throughput image. With our newly created DD mathematical model, which has been validated by computer simulations, global analysis of gene expression by DD technology is no longer a shot in the dark. After identifying the "rate-limiting" factors that contribute to the "noise" level of DD method, we have optimized the DD process with a new platform that incorporates fluorescent digital readout and automated liquid handling. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. We are using this newly integrated FDD technology to conduct a systematic and comprehensive screening for p53 tumor-suppressor gene targets.

Key Words: Differential display; p53 target genes; apoptosis. 1. Introduction

Cancer is a disease state caused by multiple genetic alterations, which lead to unregulated cell proliferation. The most frequently mutated gene among all genes known to be involved in human cancers is the tumor suppressor p53. A major outcome of such mutations is inactivation of the biochemical and biological functions of the wild-type (wt) p53 protein. Among the biological effects elicited by wtp53, the best documented are cell-cycle arrest and programmed

From: Methods in Molecular Biology, vol. 317: Differential Display Methods and Protocols Edited by: P. Liang, J. D. Meade, and A. B. Pardee © Humana Press Inc., Totowa, NJ

cell death (apoptosis). p53 is a transcription factor that can mediate many downstream effects by the activation or repression of target genes. The tumor suppressor p53 is activated by a variety of cellular stresses such as heat shock, hypoxia, osmotic shock, and DNA damage (e.g., UV), which in turn leads to growth arrest and/or apoptosis. Apoptosis is likely to be the most important function of p53 in suppressing tumor formation. However, the mechanism by which p53 actually induces apoptosis remains to be determined. p53 can induce expression of proteins that target both the mitochondrial and the death-receptor-induced apoptotic pathways. Presently, particular interest has focused on identifying target genes that mediate p53-induced apoptosis, because the induction of programmed cell death appears to be critical component of p53-medi-ated tumor suppression and because of the therapeutic potential of reactivation of this response in tumors. Recently, several p53 target genes were reported, which appear to contribute to p53-dependent apoptosis pathways (1-4). Remarkably, many of these p53 target genes were found by differential display (DD) method (4). However, the identification of additional, if not all, p53 target genes is of great importance, because this could provide the missing link between p53-mediated apoptosis and tumor suppression.

In this chapter, we present the procedure of DD and also discuss some critical factors affecting the accuracy of the method. DD methodology was invented in 1992 (5). Traditionally, DD is based on 33P radioactive labeling of cDNA bands. This is the most commonly used DD technology, because of its sensitivity, simplicity, versatility, and reproducibility. Since its creation, numerous differentially expressed genes have successfully been identified in diverse biological fields such as cancer research, developmental biology, neuroscience, plant physiology, and many other areas. Recently, a very similarly sensitive DD method was established by fluorescent labeling (6). This is the first nonradioactive DD system with sensitivity equivalent to that of the 33P isotopic labeling method. Fluorescent labeling utilizes automation, which can greatly increase the throughput and accuracy of DD.

The general strategy of fluorescence differential display (FDD), which is very similar to radioactive DD, is outlined in Fig. 1. Total RNA is used for DD comparison in gene expression. Removal of all chromosomal DNA from the RNA samples with DNase I is essential before carrying out DD, so that interfering bands amplified from genomic DNA can be unmasked. The principle of the DD and FDD method is to detect differential gene expression patterns by reverse transcription polymerase chain reaction (RT-PCR) using one of the three individual one-base anchored oligo-dT primers that anneals to the beginning of a subpopulation of the poly(A) tails of mRNAs. The anchored oligo-dT (H-T11V) primers consist of 11 Ts (T11) with a 5' HindIII (AAGCTT) site, plus one additional 3' base V (V may be dG, dA, or dC), which provides

Fig. 1. Schematic representation of fluorescent differential display (FDD) method. (Illustration courtesy of GenHunter.)

specificity. For FDD, fluorescent (Rhodamine, red)-labeled anchored R-H-T11V primers are combined with various arbitrary primers (13mer, also containing a 5' HindIII site, H-AP primers) in the PCR steps. The amplified PCR

products up to 700 bp can be separated on a denaturing polyacrylamide gel. FDD image can be obtained using a fluorescent laser scanner. Side-by-side comparisons of cDNA patterns between or among relevant RNA samples reveal differences in gene expression. The cDNA fragments of interest can be retrieved from the gel, purified, and reamplified with the same set of primers (but without fluorescent labeling of H-TnV primer) under the same PCR conditions as in the initial FDD-PCR reactions. For further molecular characterization, the obtained reamplified PCR fragments can be sequenced directly with arbitrary primers, or cloned first before sequencing if a reamplified cDNA band contains more than one mRNA species. DNA sequence analysis of these cDNA fragments by Blast Search of the Genbank (http://www.ncbi.nlm. nih.gov/ BLAST/) may provide bioinformatics information on whether a gene identified by DD is known, homologous to known, or novel.

The final step of the DD procedure is to confirm the differential expression of the mRNA identified by DD through Northern blot analysis using the same RNA samples as used for DD screenings. Such analysis provides not only confirmation by a method independent of DD, but also information about the size and relative abundance of the gene. After confirmation by Northern blot, the cloned cDNA probe can be used to screen a cDNA library for full-length clones, which can be used for functional characterization of the gene.

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