Direct comparison of the specificity of gene silencing using antisense oligonucleotides and RNAi.
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 gene 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.
Bilanges
Benoit
B
Cancer Research Institute, University of California, San Francisco, CA 94115, USA.
Stokoe
David
D
D064413:3-Phosphoinositide-Dependent Protein Kinases; D017209:Apoptosis; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D020869:Gene Expression Profiling; D020868:Gene Silencing / Q000502:physiology*; D006801:Humans; D046228:Microarray Analysis; D016376:Oligonucleotides, Antisense / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering / Q000378:metabolism*; D013997:Time Factors
D016376:Oligonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt