Journal
BIOCHEMICAL JOURNAL
Volume 388, Issue -, Pages 573-583Publisher
PORTLAND PRESS LTD
DOI: 10.1042/BJ20041956
Keywords
antisense oligonucleotide (ASO); gene silencing; microarray analysis; 3-phosphoinositide-dependent kinase 1 (PDK1); RNA interference (RNAi); small interfering RNA (siRNA)
Categories
Funding
- NCI NIH HHS [R01CA79548, P50 CA97257, P01 CA064602, P01 CA64602, P50 CA097257] Funding Source: Medline
Ask authors/readers for more resources
RNAi (RNA interference) and ASO (antisense oligonucleotide) technologies are the most commonly used approaches for silencing gene expression. However, the specificity of such powerful tools is an important factor to correctly interpret the biological consequences of acne silencing. In the present study, we examined the effects of acute loss of Ser/Thr kinase PDK1 (3-phosphoinositide-dependent kinase 1) expression using ASO and RNAi, and compared. for the first time, these two techniques using Affymetrix microarrays. We show that both ASO- and siRNA (small interfering RNA)-mediated knock-down of PDK1 expression strongly inhibited cell proliferation, although by different mechanisms, thereby questioning the specificity of these reagents. Using microarray analysis, we characterized the specificity of the ASO- and siRNA-mediated gene silencing of PDK1 by examining expression profiles 48 and 72 h following oligonucleotide transfection. At 48 h, a PDK1-dependent pattern of gene alterations was detectable, despite a large number of non-specific changes due to transfection of control nucleic acids. These non-specific alterations became more apparent at the 72 h time point, and obscured any PDK1-specific pattern. This study underscores the importance of defining appropriate control ASOs and siRNAs, using multiple oligonucleotides for each target and preferably short time points following transfection to avoid misinterpretation of the phenotype observed.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available