Gene silencing in mammalian cells through RNA interference (RNAi) has become an invaluable tool for the study of biological processes. RNAi is mediated through the action of small ∼21 nucleotide duplex RNAs, known as small interfering RNAs (siRNAs), which form part of an endogenous enzyme complex, termed the RNA-induced silencing complex (RISC). More specifically, a single strand of the siRNA duplex is loaded into RISC where it provides stringent guidance for the catalytic cleavage of complementary mRNA transcripts. We are employing RNAi for the exploration of cancer-associated pathways including the validation of anti-cancer targets, the potential identification of new targets, the development of novel model systems, and the elucidation/validation of integral components of cancer related phenotypes. Towards this end, we have developed an automated synthetic siRNA “reverse transfection” protocol on a 96 well platform (BioRobot® 8000, Qiagen Inc.). By utilizing the silencing of a siRNA targeted fluorescent marker gene as a measure of RNAi efficacy, we have optimized this protocol to yield a significant degree of RNAi with negligable cytotoxicity in a variety of cell lines. Currently, we are evaluating RNAi against approximately 350 cancer related gene targets. At least two synthetic siRNAs are being employed per target. These siRNAs were designed through the use of a proprietary algorithm (Qiagen Inc.) in an effort to improve the probability of successful gene silencing. siRNA sequences obtained from this algorithm were subjected to additional bioinformatic analysis in an attempt to further reduce potential off-target interactions. Using a branched DNA based RNA assay (Genospectra Inc.) to measure the levels of targeted transcripts, the efficacy of many of these RNAi effectors has been evaluated. On average, greater than 50% of these siRNAs have exhibited an appreciable degree of gene silencing at the RNA level in the colon cell line HCT-116. Where successful, some of these siRNAs have been further evaluated for their downstream affects on targeted proteins. For example, evaluation of a siRNA targeting ABCB1, a gene associated with multiple drug resistant cancers, in NCI/ADR-RES cells has been found to reduce target transcript levels by more than 70% and has exhibited similar results on a protein level. Furthermore, treatment of this drug resistant cell line with ABCB1 siRNA mitigated chemotherapeutic drug resistance. This type of large-scale analysis should facilitate the improved design of RNAi effector molecules, and provide insight into the cancer related roles of specific genes, including their evaluation as potential molecular targets.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]