Direct comparison of the specificity of gene silencing using antisense oligonucleotides and RNAi
Direct comparison of the specificity of gene silencing using antisense oligonucleotides and RNAi
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DOI：
10.1042/bj20041956
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发表时间：
2005-06-01
影响因子：
4.1
通讯作者：
Stokoe, D
中科院分区：
文献类型：
--
作者：
Bilanges, B;Stokoe, D
RNAi （RNA干扰）和ASO（反义寡核苷酸）技术是最常用的沉默基因表达的方法。然而，这种强大工具的特异性是正确解释痤疮沉默的生物学后果的重要因素。在本研究中，我们使用ASO和RNAi检测了丝氨酸/苏氨酸激酶PDK1（3-磷酸肌苷依赖性激酶1）表达急性丧失的影响，并进行了比较。这两种技术首次使用Affymetrix微阵列。我们发现ASO-和siRNA（小干扰RNA）介导的PDK1表达下调都能强烈抑制细胞增殖，尽管机制不同，从而质疑这些试剂的特异性。利用微阵列分析，我们通过检测寡核苷酸转染后48和72小时的表达谱，表征了ASO和sirna介导的PDK1基因沉默的特异性。在48小时，尽管由于转染对照核酸而产生大量非特异性变化，但仍可检测到pdk1依赖的基因改变模式。这些非特异性改变在72h时间点变得更加明显，并且掩盖了任何pdk1特异性模式。这项研究强调了定义适当的对照ASOs和sirna的重要性，对每个靶标使用多个寡核苷酸，最好在转染后的短时间点使用，以避免对观察到的表型产生误解。
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.