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Using Real-time qPCR to Evaluate RNAi-mediated Gene Silencing
Sponsored,vendor-submitted protocol   Sponsored by Bio-Rad Laboratories    Published in BioTechniques Protocol Guide 2009 (p.47) DOI: 10.2144/000113006

Introduction

Neuroblastoma is a childhood cancer that is derived from precursor cells of the adrenosympathetic system, originating in the adrenal medulla or sympathetic ganglia. To gain insight into the mechanism of action of the MYCN gene in neuroblastoma pathogenesis, we used RNA interference (RNAi) to suppress MYCN expression. The MYCN gene was transiently suppressed using 27-mer siLentMer™*

*These siLentMers were validated in Dr. Vandesompele's laboratory. Dicersubstrate small interfering RNA (siRNA) duplexes (Bio-Rad Laboratories, Inc.), which are generally considered more effective at gene silencing than their corresponding traditional 21-mer siRNAs (1). The CFX96™ real-time PCR detection system (Bio-Rad Laboratories, Inc.) was used to monitor silencing efficiency and assess functional effects of RNAi-mediated knockdown of MYCN. Assessment of siRNA Silencing Efficiency Using Real-time Quantitative PCR (rt-qPCR) in Cells Treated with Anti-MYCN siLentMer siRNA Duplexes

Human IMR-32 neuroblastoma cells were transfected with two anti-MYCN siLentMer siRNA duplexes and the level of MYCN transcript was determined 48 h posttransfection by rt-qPCR using five different primer pairs (Figure 1A). Results summarized in Figure 1 show variable levels of MYCN silencing observed with the different primer pairs (Figure 1B). These results indicate the importance of primer location for evaluation of siRNA silencing efficiency, consistent with a previously published study (2). The target mRNA sequence is cleaved by the RNA-induced silencing complex near the center of the region complementary to the guiding siRNA (3) and complete nucleolytic degradation of the resulting fragments does not always occur. Therefore, using primers that do not span the siRNA target sequence may result in an underestimation of siRNA silencing efficiency.


Figure 1. Importance of primer location for rt-qPCR assessment of siRNA silencing efficiency. (Click to enlarge)


Conclusion

rt-qPCR is the method of choice for accurate, sensitive, and specific quantitation of nucleic acid sequences. It provides a convenient and reliable tool for evaluating knockdown efficiency and functional effects of RNAi-mediated gene silencing, as demonstrated by the use of this tool for monitoring knockdown of MYCN, a key gene in neuroblastoma pathogenesis. Successful application of rt-qPCR requires careful attention to all of the steps in the assay workflow, including primer design and evaluation, template preparation, normalization strategy, and data analysis. Correspondence

Address correspondence to Bio-Rad Laboratories, Inc., Life Science Group, 2000 Alfred Nobel Drive, Hercules, CA 94547, USA; Tel.: 1-800-4BIORAD (1-800-424-6723); www.discover.bio-rad.com. For Product Technical Support: [email protected] For Literature Request: [email protected]
References
1.) Kim, D.H.. 2005. Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol. 23:222-226.

2.) Shepard, A.R.. 2005. Importance of quantitative PCR primer location for short interfering RNA efficacy determination. Anal. Biochem. 344:287-288.

3.) Elbashir, S.M.. 2001. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15:188-200.



*These siLentMers were validated in Dr. Vandesompele's laboratory.

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