Identification of differential gene expression profiles in rat cortical cells exposed to the neuroactive agents trimethylolpropane phosphate and bicuculline

Recent technological advancements in microfabrication combined with the rap id acquisition of full genome sequence data have led to the development of DNA arrays that have the capacity to monitor the expression levels of thous ands of genes simultaneously. The development of this technology enables th e use of functional genomics approaches to identify molecular markers assoc iated with Cellular responsiveness to cytotoxic exposures. Databases contai ning unique cell-response profiles associated with specific toxicants or cl asses of toxicants can then be used in conjunction with cell-based biosenso r platforms for environmental surveillance and toxicological assessment. An important issue that must be addressed, however, is whether DNA arrays can be used to identify transient gene modulation events in a reproducible man ner. To address this issue, we utilized a primary embryonic rat (day 18) co rtical cell model system and examined the RNA of both chemically treated an d untreated cells using radioisotope-labeled cDNA probes and commercially a vailable nylon membrane arrays. Using this approach, we examined experiment al variability, basal gene expression variability, the occurrence of false positives, and the reproducibility of gene expression profiles obtained aft er chemical exposure. Minimal differences in gene modulation were observed between RNA samples from independently cultured cortical cells when array e xperiments were conducted in parallel (Pearson correlation coefficient for gene intensities = 0.98). In contrast, significant differences in gene expr ession were observed between array experiments conducted at different times with an identical RNA source (Pearson correlation coefficient for gene int ensities = 0.91). Our results suggest the effect of basal gene activity dif ferences in independently isolated cell cultures is negligible and that exp erimental variability possibly associated with the handling of RNA samples, differences in reverse transcription efficiency, hybridization, and/or sig nal acquisition are the primary contributors to variability in measurements . Using cDNA array analysis of unexposed cells from three independent cell culture preparations, we calculated false positive gene modulation events a s a function of the threshold \ log(2) R \ > 1.0. The number of false posit ives using this criteria was 1 - 10 gene/ESTs/5109 actively transcribed gen e/ESTs represented on the array. Using three independent replicate experime nts of untreated cortical cell cultures, we determined that a threshold cri terion of \ log(2) R \ > 0.63 for triplicate experiments would reduce the e xpected number of false positives in our experiments to less than one. Usin g this criterion, reproducible gene expression profiles were identified in cortical cells exposed to the neuroactive agents trimethylolpropane phospha te and bicuculline. (C) 2001 Elsevier Science B.V. All rights reserved.