Functional studies on transfected cell microarray analysed by linear regression modelling.
Transfected cell microarray is a promising method for accelerating the functional exploration of the genome, giving information about protein function in the living cell. The microarrays consist of clusters of cells (spots) overexpressing or silencing a particular gene product. The subsequent analysis of the phenotypic consequences of such perturbations can then be detected using cell-based assays. The focus in the present study was to establish an experimental design and a robust analysis approach for fluorescence intensity data, and to address the use of replicates for studying regulation of gene expression with varying complexity and effect size. Our analysis pipeline includes measurement of fluorescence intensities, normalization strategies using negative control spots and internal control plasmids, and linear regression (ANOVA) modelling for estimating biological effects and calculating P-values for comparisons of interests. Our results show the potential of transfected cell microarrays in studying complex regulation of gene expression by enabling measurement of biological responses in cells with overexpression and downregulation of specific gene products, combined with the possibility of assaying the effects of external stimuli. Simulation experiments show that transfected cell microarrays can be used to reliably detect even quantitatively minor biological effects by including several technical and experimental replicates.
Fjeldbo
Christina Saeten
CS
Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway. christina.saten@ntnu.no
Misund
Kristine
K
Günther
Clara-Cecilie
CC
Langaas
Mette
M
Steigedal
Tonje Strømmen
TS
Thommesen
Liv
L
Laegreid
Astrid
A
Bruland
Torunn
T
D002460:Cell Line; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016014:Linear Models; D016328:NF-kappa B / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D012097:Repressor Proteins / Q000378:metabolism; D046888:Tissue Array Analysis / Q000379:methods*; D015533:Transcriptional Activation; D014162:Transfection
D005456:Fluorescent Dyes; D016328:NF-kappa B; D012097:Repressor Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins