Small interfering siRNA (siRNA) is a class of 20-25 nucleotide double-stranded RNA molecules, which interfere with the expression of specific genes with complementary sequences, leading to RNA interference (RNAi). siRNAs have become a powerful tool for studying the function of genes.
RNAi can be induced artificially in mammalian cells by using various methods. For long-term silencing of a specific gene, cells may be transfected with an appropriate vector (e.g. a plasmid) expressing a precursor siRNA sequence, which may be processed into a functional siRNA within the cells. For short-term silencing (max 72 hours), cells may be transfected with synthetic siRNAs.
A potent and specific siRNA design is a pre-requisite to obtain a satisfactory level of mRNA knock down, without modifying the expression of unintended targets. Therefore, some companies have developed proprietary tools for designing and screening siRNA sequences.
I apply the RNAi technique to study the role of transcription factors in the regulation of specific genes. I successfully transfected synthetic pre-designed siRNAs (Qiagen) for knocking-down the expression of various genes in primary cultures of rat aortic endothelial cells and human skin fibroblasts, using HiPerfect Transfetion Reagent (Qiagen).
Pre-designed synthetic siRNAs for silencing the expression of a gene of interest present the main advantages of being 1) ready to use; the experiment may be started soon after obtaining the optimal transfection conditions (i.e. the optimal transfection reagent/siRNA ratio); 2) relatively inexpensive; in fact, only the transfection reagent and the sequences have to be purchased; 3) used in high throughput applications, if desired. The main disadvantage is that synthetic siRNA can only be used for transient transfections in mammalian cells, which may be a problem for experiments conducted in rapid dividing cells or requiring a long-term analysis of the effects produced.
I began to work with siRNA about 3 years ago. I needed to silence two transcription factors in rat cells. Since at that time, no pre-designed sequences were available for rat genes, I used the custom design and synthesis service of Qiagen, called HP Flexible siRNA Design. I sent the sequence information of the target genes (i e.RefSeq entries, GenBank accession number), and I received my siRNAs together with information about the targeted sequence, the siRNA sequence, and the handling protocol. Maybe I was very lucky, but all the siRNAs I ordered worked really well. Recently, pre-designed siRNAs for all human, mouse and rat are available (HP GenomeWide siRNA, Qiagen), but information of the sequences is not provided. For a smaller number of genes, Qiagen also offers predesigned and functionally validated siRNAs which have been tested by real-time RT-PCR to provide at least 70% knockdown (HP Validated siRNA, Qiagen); for HP Validated siRNA, the sequence is provided. I used HP Validated siRNAs to successfully knock down the expression of transcription factors in primary human skin fibroblasts. I obtained a 70%-80% decrease of expression of the targeted genes, without measurable off-target effects (as determined by cross comparing the effects exerted on the various genes analyzed).
I would recommend the use of synthetic siRNA for short term analyisis of the knock down of genes in mammalian cells. Since the sequence of HP Validated siRNA (Qiagen) is provided by the company, I would recommend it as the pre-designed synthetic siRNA of choice for knocking down the expression of genes in rat, mouse and human cells.