Critical to the work of GSS and the CCR RNAi initiative has been the utilization and development of new technologies and assays. RNA analysis: We have now fully assessed and optimized for our use a commercial RNA assay (""""""""Quantigene"""""""", Genopsectra Inc.) that allows us to conduct RNAi analysis at an RNA level in relatively high-throughput. We have now utilized the """"""""Quantigene"""""""" assay to analyze RNAi for over 100 human genes. Protein analysis: During 2005 we have developed in collaboration with Dr. Paul Goldsmith and Dr. John Weinstein a quantitative approach to RNAI analysis at a protein level. This Western blot based assay system is giving highly reproducible results. Cell lines and transfection optimization: Critical to developing a screening system to validating RNAi resources has been the determination of the most suitable cell line(s) in which to conduct this work. We routinely conducted most RNAi analysis in either a human colon cancer cell line HCT-116 or a human breast cancer cell line MDA-MB-231. We have, though also adapted our transfection protocol to over 10 additional cell lines. In addition, a significant part of this procedure has automated to reduce error and significantly enhance reproducibility. Functional analysis: We are now increasing our commitment to aligning the most suitable functional assays with RNAi analysis and this will become an increasingly important part of our work over 2006.
| Martin, Scott E; Jones, Tamara L; Thomas, Cheryl L et al. (2007) Multiplexing siRNAs to compress RNAi-based screen size in human cells. Nucleic Acids Res 35:e57 |
| Martin, Scott E; Caplen, Natasha J (2007) Applications of RNA interference in mammalian systems. Annu Rev Genomics Hum Genet 8:81-108 |
| Martin, Scott E; Caplen, Natasha J (2006) Mismatched siRNAs downregulate mRNAs as a function of target site location. FEBS Lett 580:3694-8 |
| Ludwig, Joseph A; Szakacs, Gergely; Martin, Scott E et al. (2006) Selective toxicity of NSC73306 in MDR1-positive cells as a new strategy to circumvent multidrug resistance in cancer. Cancer Res 66:4808-15 |
| Dombroski, Derek; Houghtling, Richard A; Labno, Christine M et al. (2005) Kinase-independent functions for Itk in TCR-induced regulation of Vav and the actin cytoskeleton. J Immunol 174:1385-92 |
| Huppi, Konrad; Martin, Scott E; Caplen, Natasha J (2005) Defining and assaying RNAi in mammalian cells. Mol Cell 17:1-10 |
