HT RNAi Viability Screen in DU145 Prostate Cancer Cells
The goal of the following HT RNAi Viability screen was to identify genes and corresponding androgen-dependent and independent signaling pathways which are critical for viability of prostate cells but are not essential for cells of other tissue types. A prostate-specific 7K shRNA/2K mRNA pooled lentiviral bar-coded tet-regulated shRNA library was used to infect DU145 prostate cancer cells, and cells were grown for 5 days in the presence of puromycin. Infected cells were split into a control pool (no doxycycline treatment) and a pool of cells which was treated with doxycycline (5 ug/ml) to activate tet-dependent expression of shRNAs. Surviving –dox and +dox cells were collected, and genomic DNA was purified after two weeks of growth. As a negative control, genomic DNA from the cells not infected with 7K shRNA library was used.
To identify the cytotoxic shRNAs which are responsible for reduced cell viability, the entire pool of bar-codes from genomic DNA was amplified via two rounds of PCR with vector-specific and insert-specific primers and representation of bar-codes in pools of control and dox-induced cells was analyzed by HT sequencing. A similar RNAi viability screen was successfully implemented for diffuse large B-lymphoma cell lines by Staudt’s group[1], and we used their guidelines for our experiments.
This study was made possible by NIH NCI Grant # 1R43CA134062-01, entitled "Viability Pathway Models in Prostate Cancer Cells."
View our Frequently Asked Questions (FAQs) page for detailed information on our pooled lentiviral shRNA libraries. For relevant references and literature, visit the Articles page under Resources. Visit the HT RNAi TGF-β Screen comparison data page for additional functional data.
- Ngo, V.N., Davis, R.E., Lamy, L., Yu, X., Zhao, H., Lenz., Lam, L.T., Dave, S., Yang, L., Powell, J. and Staudt, L.M. A loss-of-function RNA interference screen for molecular targets in cancer. Nature (2006) 441: 106-110.
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