Bioinformatic analysis of primary endothelial cell gene array data illustrated by the analysis of transcriptome changes in endothelial cells exposed to VEGF-A and PlGF.
We recently published a review in this journal describing the design, hybridisation and basic data processing required to use gene arrays to investigate vascular biology (Evans et al. Angiogenesis 2003; 6: 93-104). Here, we build on this review by describing a set of powerful and robust methods for the analysis and interpretation of gene array data derived from primary vascular cell cultures. First, we describe the evaluation of transcriptome heterogeneity between primary cultures derived from different individuals, and estimation of the false discovery rate introduced by this heterogeneity and by experimental noise. Then, we discuss the appropriate use of Bayesian t-tests, clustering and independent component analysis to mine the data. We illustrate these principles by analysis of a previously unpublished set of gene array data in which human umbilical vein endothelial cells (HUVEC) cultured in either rich or low-serum media were exposed to vascular endothelial growth factor (VEGF)-A165 or placental growth factor (PlGF)-1(131). We have used Affymetrix U95A gene arrays to map the effects of these factors on the HUVEC transcriptome. These experiments followed a paired design and were biologically replicated three times. In addition, one experiment was repeated using serial analysis of gene expression (SAGE). In contrast to some previous studies, we found that VEGF-A and PlGF consistently regulated only small, non-overlapping and culture media-dependant sets of HUVEC transcripts, despite causing significant cell biological changes.
Schoenfeld
Jonathan
J
Department of Pathology, Cambridge University, Cambridge, UK.
Lessan
Khashayar
K
Johnson
Nicola A
NA
Charnock-Jones
D Stephen
DS
Evans
Amanda
A
Vourvouhaki
Ekaterini
E
Scott
Laurie
L
Stephens
Richard
R
Freeman
Tom C
TC
Saidi
Samir A
SA
Tom
Brian
B
Weston
Gareth C
GC
Rogers
Peter
P
Smith
Stephen K
SK
Print
Cristin G
CG
D002478:Cells, Cultured; D019295:Computational Biology; D003470:Culture Media / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D000072483:Placenta Growth Factor; D016133:Polymerase Chain Reaction; D011257:Pregnancy Proteins / Q000494:pharmacology*; D011506:Proteins / Q000235:genetics; D015203:Reproducibility of Results; D014158:Transcription, Genetic / Q000187:drug effects*; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
D003470:Culture Media; C000608036:PGF protein, human; D011257:Pregnancy Proteins; D011506:Proteins; D042461:Vascular Endothelial Growth Factor A; D000072483:Placenta Growth Factor