Optimization of comparative expressed sequence hybridization for genome-wide expression profiling at chromosome level

Abstract

Comparative expressed sequence hybridization (CESH) has recently been developed for global expression profiling at chromosome level. To improve its specificity and sensitivity, we examined the effects of cDNA amplification and labeling methods on CESH profiles, using a gastric cancer cell line, Kato III, and compared the CESH profiles to cDNA microarray and reverse transcriptase–polymerase chain reaction (RT-PCR) data. CESH results were scarcely affected by the amplification process, either at the RNA level with T7 polymerase or at the cDNA level with degenerate oligonucleotide-primed PCR (DOP-PCR). The labeling method, however, did remarkably affect the CESH results; false positive shifts of the test/reference ratio (T/R) were not detected in self-matched CESH with pre-cDNA labeling and random priming labeling of cDNA but were consistently seen with DOP-PCR labeling in 11 chromosomes. The use of cDNA deriving from mRNA either with pre-cDNA or random priming labeling gave results of higher detection sensitivity for regions of up- or downregulated expression and higher concordance with the microarray and RT-PCR data in the corresponding regions than with conventional CESH. This modification of CESH with random priming labeling was found feasible by its application to Kato III cells with and without 5-aza-2′-deoxycytidine treatment; the regions identified as epigenetically silenced included genes that were reportedly methylated in Kato III.

Introduction

The gene expression pattern is primarily determined by genomic constitution [1] and is epigenetically regulated temporally and spatially by tissue environment and cell differentiation programs [2]. Recent advances in array technology have enabled analysis of expression of thousands of genes from a single sample simultaneously [3]. Such a high-throughput analysis of global gene expression has contributed remarkably to reappraisal of tumor classification [4], [5]. Interest has often been focused on which genes are up- or downregulated [6], [7], whereas how the up- or downregulated genes are distributed on chromosomes remained largely unexplored until recently. From expression microarray data, recent bioinformatics advances enable mapping gene expression on chromosomes [8] and identification of clusters of up- or downregulated genes [9], [10].

Global expression profiling at chromosome level was demonstrated recently with comparative expressed sequence hybridization (CESH) [11], [12], [13], [14], which is based on the same principle as comparative genomic hybridization (CGH) [15] but using cDNA instead of genomic DNA. Though the resolution of CESH is lower, its efficiency is higher than that of expression microarray. CESH data are free from the bias caused by target gene selection [12]. However, methodological aspects of CESH have not been reappraised critically so far.

It was recently shown that application of degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) [16] to probe amplification scarcely affected the sensitivity and specificity of CGH [17]. In the present study, using a gastric cancer cell line, we assessed whether this is also the case for CESH and whether another probe amplification with T7 RNA polymerase affects the CESH profile. The previous reports of CESH [11], [12], [13], [14] used DOP-PCR as the labeling as well as the amplification method; however, the reliability of DOP-PCR labeling remains to be established. We therefore compared CESH using DOP-PCR labeling with CESH using random priming (RP) labeling of cDNA or pre-cDNA labeling during reverse transcription. We also compared CESH profiles to the expression pattern detected by cDNA microarray and to the expression of specified genes demonstrated by real-time RT-PCR.

Based on these data, we have proposed a better method of CESH for global gene expression analysis, of which the feasibility was tested by applying it to the detection of epigenetically silenced chromosomal regions with 5-aza-2′-deoxycytidine (5-Aza-dC) treatment.