Insights into effective RNAi gained from large-scale siRNA validation screening (original) (raw)
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Validation of Short Interfering RNA Knockdowns by Quantitative Real-Time PCR
RNA interference (RNAi) is a natural mechanism, that is triggered by the introduction of double-stranded RNA into a cell. The long double-stranded RNA is then processed into short interfering RNA (siRNA) that mediates sequence-specific degradation of homo-logous transcripts. This phenomenon can be exploited to experimentally trigger RNAi and downregulate gene expression by transfecting mammalian cells with synthetic siRNA. Thus, siRNAs can be designed to specifically silence the expression of genes bearing a particular target sequence. In this chapter, we present methods and procedures for validating the effects of siRNA-based gene silencing on target gene expression. To illustrate our approach, we use examples from our analysis of a Cancer Gene Library of 278 siRNAs targeting 139 classic oncogenes and tumor suppressor genes (Qiagen Inc., Germantown, MD). Specifically, this library was used for high-throughput RNAi phenotype analysis followed by gene expression analysis to validate gene silencing for siRNA that produced a phenotype. Methods and protocols are presented that illustrate how sequence-specific gene silencing of effective siRNAs are analyzed and validated by quantitative real-time PCR assays to measure the extent of target gene silencing, as well as effects on various gene expression end points.
Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing
Gene, 2007
RNA interference (RNAi)-mediated gene silencing has become a valuable tool for functional studies, reverse genomics, and drug discoveries. One major challenge of using RNAi is to identify the most effective short interfering RNAs (siRNAs) sites of a given gene. Although several published bioinformatic prediction models have proven useful, the process to select and validate optimal siRNA sites for a given gene remains empirical and laborious. Here, we developed a fluorescence-based selection system using a retroviral vector backbone, namely pSOS, which was based on the premise that candidate siRNAs would knockdown the chimeric transcript between GFP and target gene. The expression of siRNA was driven by the opposing convergent H1 and U6 promoters. This configuration simplifies the cloning of duplex siRNA oligonucleotide cassettes. We demonstrated that GFP signal reduction was closely correlated with siRNA knockdown efficiency of human β-catenin, as well as with the inhibition of β-catenin/Tcf4 signaling activity. The pSOS should not only facilitate the selection and validation of candidate siRNA sites, but also provide efficient delivery tools of siRNAs via viral vectors in mammalian cells. Thus, the pSOS system represents an efficient and user-friendly strategy to select and validate siRNA target sites.
DSIR: Assessing the Design of Highly Potent siRNA by Testing a Set of Cancer-Relevant Target Genes
PLoS ONE, 2012
Chemically synthesized small interfering RNA (siRNA) is a widespread molecular tool used to knock down genes in mammalian cells. However, designing potent siRNA remains challenging. Among tools predicting siRNA efficacy, very few have been validated on endogenous targets in realistic experimental conditions. We previously described a tool to assist efficient siRNA design (DSIR, Designer of siRNA), which focuses on intrinsic features of the siRNA sequence. Here, we evaluated DSIR's performance by systematically investigating the potency of the siRNA it designs to target ten cancerrelated genes. mRNA knockdown was measured by quantitative RT-PCR in cell-based assays, revealing that over 60% of siRNA sequences designed by DSIR silenced their target genes by at least 70%. Silencing efficacy was sustained even when low siRNA concentrations were used. This systematic analysis revealed in particular that, for a subset of genes, the efficiency of siRNA constructs significantly increases when the sequence is located closer to the 59-end of the target gene coding sequence, suggesting the distance to the 59-end as a new feature for siRNA potency prediction. A new version of DSIR incorporating these new findings, as well as the list of validated siRNA against the tested cancer genes, has been made available on the web (http://biodev.extra.cea.fr/DSIR).
No independent cross-validation of success rate for studies utilizing small interfering RNA (siRNA) for gene silencing has been completed before. To assess the influence of experimental parameters like cell line, transfection technique, validation method, and type of control, we have to validate these in a large set of studies. We utilized gene chip data published for siRNA experiments to assess success rate and to compare methods used in these experiments. We searched NCBI GEO for samples with whole transcriptome analysis before and after gene silencing and evaluated the efficiency for the target and off-target genes using the array-based expression data. Wilcoxon signed-rank test was used to assess silencing efficacy and Kruskal–Wallis tests and Spearman rank correlation were used to evaluate study parameters. All together 1,643 samples representing 429 experiments published in 207 studies were evaluated. The fold change (FC) of down-regulation of the target gene was above 0.7 in 18.5% and was above 0.5 in 38.7% of experiments. Silencing efficiency was lowest in MCF7 and highest in SW480 cells (FC = 0.59 and FC = 0.30, respectively, P = 9.3E−06). Studies utilizing Western blot for validation performed better than those with quantitative polymerase chain reaction (qPCR) or microarray (FC = 0.43, FC = 0.47, and FC = 0.55, respectively, P = 2.8E−04). There was no correlation between type of control, transfection method, publication year, and silencing efficiency. Although gene silencing is a robust feature successfully cross-validated in the majority of experiments, efficiency remained insufficient in a significant proportion of studies. Selection of cell line model and validation method had the highest influence on silencing proficiency. Molecular Therapy—Nucleic Acids (2016) 5, e366; doi:10.1038/mtna.2016.66; published online 27 September 2016 Subject Category: siRNA, shRNAs and miRNAS
Global Gene Expression Effects of siRNA-based Gene Silencing
Small interfering RNA (siRNA) is a powerful functional genomic tool used to silence the expression of targeted genes. A genome-wide view of this phenomenon has allowed identification (in addition to specific gene knockdown) of both sequencespecific "off-target" effects (Jackson, 2003) and sequence non-specific toxicity affects . In this study we have used Agilent's in situ synthesized 60-mer oligonucleotide microarrays to systematically study siRNA-based off-targeting and toxic effects siRNAs are most often introduced into cells using lipid-mediated transfection. The large-scale expression signature represents a combination of both transfection and siRNA-specific effects. We have evaluated the degree of the cellular response due to siRNAbased effects and transfection-based artifacts. A previously reported siRNA-based induction of an interferon response is here observed only in the background of an invasive or toxic transfection protocol and can be sequence-dependent. Optimization of transfection conditions (minimization of lipid-based toxicity) or use of alternative delivery technologies can minimize siRNA-based toxicity. In conclusion, global expression effects of a given siRNA are minimal and may be controlled by careful sequence selection and experimental design.
Selection of highly efficient small interference RNA (SiRNA) targeting mammalian genes
2013
RNAi is the method of silencing the expression of targeted genes. RNAi applications include gene function analysis and target validation. Designing highly efficient small interference RNA (siRNA) sequence with maximum target specificity for mammalian RNAi is one of important topics in recent years. In this work, a statistical analysis of the information for a large number (3734) of siRNA presented in the database available on the internet is done. This is to improve the design of efficient siRNA molecules. The (3734) siRNAs are classified according to their efficiency to three groups (high efficient, moderate efficient and low efficient). Thirteen properties (positional and thermodynamics) are identified in the high efficient group in the primary statistical study. In the final statistical study, the average weight of each identified property is calculated. A very good linear correlation was found between the average percentage efficiency and the weighted score of siRNA properties. It is found that the most important feature of highly efficient siRNA is the difference in binding energy between the 5' end and the 3' end of the anti-sense strand. The (RISC) activation step is a critical step in RNAi process where the efficiency of this process depends on the instability of the 5' end of the anti-sense strand.
Exploring the specificity and mechanisms of siRNA-mediated gene silencing in mammalian cells
2008
Complementary short interfering RNAs (siRNAs) are routinely used to knockdown gene expression. siRNAs bind to their target sequence and guide transcript cleavage and subsequent degradation. This type of silencing is associated with equivalent levels of RNA and protein knockdown. siRNA-mediated knockdown was originally thought to be highly specific. However, the downregulation of non-target mRNAs has been observed following transfection of siRNAs in human cells. Many of these RNA changes are due to siRNA binding to partially complementary sequences within nontargeted transcripts and therefore are termed "off-target" effects. To examine the mRNA:siRNA interactions important for off-target effects, we generated a panel of mRNA:siRNA combinations containing a variety of base pairing interactions in the 9 th , 10 th , and 11 th positions of two siRNA binding sites located in a reporter gene. This region was chosen because siRNA-mediated transcript cleavage occurs between the 10 th and 11 th positions of the mRNA:siRNA duplex. Approximately half of the mRNA:siRNA combinations containing mismatches in positions 9-11 resulted in a twofold or more mRNA decrease, with varying degrees of protein knockdown. mRNA and protein analysis revealed combinations for which the resulting mRNA and protein levels did not correlate. Although siRNA-mediated transcript cleavage is catalyzed by the endonuclease Argonaute 2 (Ago2), knockdown of Ago2 expression did not affect mRNA knockdown for imperfectly complementary combinations. These results indicate that off-target mRNA reductions are likely attributable to Ago2-independent degradation processes. Using the same reporter system we have also uncovered instances in which complementary siRNAs resulted in high protein/RNA knockdown ratios with dramatic protein silencing. For these particular combinations, the disparity between RNA and protein knockdown is dependent on Ago2 function. This may suggest that the sequences within atypical complementary mRNA:siRNA combinations result in unproductive cleavage by Ago2, leading to persistent binding and enhanced silencing through translational repression. Our findings demonstrate that differences within complementary target sequences can lead to differences in the type of silencing mechanisms that result. The results presented in this dissertation also provide a better understanding of how off-target effects mediate mRNA and protein knockdown of unrelated transcripts, with meaningful implications for those using siRNAs as a tool.
Biochemical and Biophysical Research Communications, 2006
Small interfering RNAs (siRNAs) have become the most powerful and widely used gene silencing reagents for reverse functional genomics and molecular therapeutics. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily dependent on the effectiveness and specificity of the RNAi targeting sequence. However, only a limited number of siRNAs is capable of inducing highly effective and sequence-specific gene silencing by RNA interference (RNAi) mechanism. In addition, the efficacy of siRNA-induced gene silencing can only be experimentally measured based on inhibition of the target gene expression. Therefore, it is important to establish a fully robust and comparative validating system for determining the efficacy of designed siRNAs. In this study, we have developed a reliable and quantitative reporter-based siRNA validation system that consists of a short synthetic DNA fragment containing an RNAi targeting sequence of interest and two expression vectors for targeting reporter and triggering siR-NA expression. The efficacy of the siRNAs is measured by their abilities to inhibit expression of the targeting reporter gene with easily quantified readouts including enhanced green fluorescence protein (EGFP) and firefly luciferase. Using fully analyzed siRNAs against human hepatitis B virus (HBV) surface antigen (HBsAg) and tumor suppressor protein p53, we have demonstrated that this system could effectively and faithfully report the efficacy of the corresponding siRNAs. In addition, we have further applied this system for screening and identification of the highly effective siRNAs that could specifically inhibit expression of mouse matrix metalloproteinase-7 (MMP-7), Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and human serine/threonine kinase AKT1. Since only a readily available short synthetic DNA fragment is needed for constructing this novel reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective siRNAs but also implicates in the use of RNAi for studying novel gene function in mammals.