The RNA interference pathway (also called RNAi) is an evolutionarily conserved physiological pathway, which plays a vital role in gene regulation, genome defense and chromatin architecture and dynamics. The functional intermediates of the RNAi pathway, small interfering RNA (also called siRNA) are highly effective genetic tools for specifically silencing the expression of a particular gene. The application of RNAi represents a major breakthrough in functional genomics. The use of siRNA methodology has revolutionized the study of gene function and regulation, led to the development of novel disease models in animals and has numerous potential diagnostic and therapeutic applications. A major challenge in siRNA research has been target-specific gene knockdown with minimum off-target gene modulation.
The BLOCK-iT™ Dicer RNAi Transfection Kit involves the generation of a pool of diced siRNA (using the Dicer enzyme) and subsequent transfection of cells with this pool of siRNA to block the expression of your gene of interest. The biggest advantage of the BLOCK-iT™ Dicer RNAi Transfection Kit is that there is no need to predesign and predetermine the target sequences for the gene. The mixture of diced siRNA ensures that there will be at least some sequences which will produce highly efficacious gene silencing. The kit is especially useful if you want to block the expression of a relatively novel and unknown gene. The kit is easy to use, has plenty of reagents and has an excellent instruction booklet equipped with detailed protocols.
Our laboratory used the BLOCK-iT™ Dicer RNAi Transfection Kit for generating siRNAs against prohibitin, a potential tumor gene, and E2F1. With the help of the Dicer Kit, we generated an siRNA pool against E2F1 and prohibitin in about 48 hours. Upon transfection of the RNAi’s in MCF-7 breast cancer cells, we observed a significant suppression of E2F1 and prohibitin levels as determined by Western blot analysis. However, our results show that the siRNAs could ablate E2F and prohibitin protein expression for only about 72 hours; after this time, the levels of the proteins start to slowly come back, which is a vital consideration for designing experiments involving RNAi.
The main disadvantage of the BLOCK-iT™ Dicer RNAi Transfection Kit is that the procedure to generate the siRNA pool is quite long and time consuming. First, the gene of interest has to be cloned in cis and trans, followed by transcription of the gene in both orientations. The BLOCK-iT™ Dicer RNAi Transfection Kit does not include any reagents for transcription (the Complete BLOCK-iT™ Dicer RNAi Transfection Kit does), so you have to provide those. The third step is the generation of the diced siRNA using the Dicer enzyme. Although, the pool of siRNA is more effective in suppressing gene expression, it is not possible to determine which sequences in the siRNA pool are actually responsible for effective gene silencing. The presence of a mixture of siRNAs also increases the danger of off-target effects in the cellular expression profile. On the other hand, chemically synthesized defined siRNA oligonucleotide sequences, which ablate expression of specific genes are available from many companies. The transfection of these siRNA oligonucleotides takes less time and produces equally efficient abrogation of gene expression. However, as I previously mentioned, the BLOCK-iT™ Dicer RNAi Transfection Kit is excellent for relatively novel and uncommon genes for which siRNAs are yet to be commercially available. I would wholeheartedly recommend it as a valuable tool for the biological scientists involved in the study of gene expression and regulation.
Piyali Dasgupta
Postdoctoral Fellow
H. Lee Moffitt Cancer Center
Drug Discovery