Pooling samples within microarray studies: a comparative analysis of rat liver transcription response to prototypical toxicants.
Combining or pooling individual samples when carrying out transcript profiling using microarrays is a fairly common means to reduce both the cost and complexity of data analysis. However, pooling does not allow for statistical comparison of changes between samples and can result in a loss of information. Because a rigorous comparison of the identified expression changes from the two approaches has not been reported, we compared the results for hepatic transcript profiles from pooled vs. individual samples. Hepatic transcript profiles from a single-dose time-course rat study in response to the prototypical toxicants clofibrate, diethylhexylphthalate, and valproic acid were evaluated. Approximately 50% more transcript expression changes were observed in the individual (statistical) analysis compared with the pooled analysis. While the majority of these changes were less than twofold in magnitude ( approximately 80%), a substantial number were greater than twofold (approximately 20%). Transcript changes unique to the individual analysis were confirmed by quantitative RT-PCR, while all the changes unique to the pooled analysis did not confirm. The individual analysis identified more hits per biological pathway than the pooled approach. Many of the transcripts identified by the individual analysis were novel findings and may contribute to a better understanding of molecular mechanisms of these compounds. Furthermore, having individual animal data provided the opportunity to correlate changes in transcript expression to phenotypes (i.e., histology) observed in toxicology studies. The two approaches were similar when clustering methods were used despite the large difference in the absolute number of transcripts changed. In summary, pooling reduced resource requirements substantially, but the individual approach enabled statistical analysis that identified more gene expression changes to evaluate mechanisms of toxicity. An individual animal approach becomes more valuable when the overall expression response is subtle and/or when associating expression data to variable phenotypic responses.
Jolly
Robert A
RA
Lilly Research Laboratories, Greenfield, Indiana 46140, USA.
Goldstein
Keith M
KM
Wei
Tao
T
Gao
Hong
H
Chen
Peining
P
Huang
Shuguang
S
Colet
Jean-Marie
JM
Ryan
Timothy P
TP
Thomas
Craig E
CE
Estrem
Shawn T
ST
D000818:Animals; D002994:Clofibrate / Q000633:toxicity; D016000:Cluster Analysis; D004051:Diethylhexyl Phthalate / Q000633:toxicity; D005227:Fatty Acids / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D008099:Liver / Q000187:drug effects* / Q000378:metabolism*; D008110:Liver Extracts / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D015233:Models, Statistical; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009929:Organ Size; D010641:Phenotype; D010802:Phylogeny; D025341:Principal Component Analysis; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014635:Valproic Acid / Q000633:toxicity
D005227:Fatty Acids; D008110:Liver Extracts; D012333:RNA, Messenger; D014635:Valproic Acid; D012313:RNA; D004051:Diethylhexyl Phthalate; D002994:Clofibrate