RJH Transfection Reagents in siRNA Library Screens
RJH Transfection Reagents in siRNA Library Screens
Background:
The structured oligonucleotide libraries allow one to screen large number of compounds for specific purposes. In biomarker discovery, library screens facilitate identification of critical mediators that are responsible for pathological conditions. In therapeutic discovery, library screens allow one to identify new chemical entities for a desired therapeutic action. Having the ability to screen large numbers of compounds in one attempt is advantageous to provide head-to-head comparisons in an expeditious manner. However, one has to rely on a reproducible procedure and delivery that are uniformly similar for each member of the library component. This is especially the case when transfection reagents are used where robust performance is expected for the delivery of each component of the library. Having to work with large libraries causes a cost burden that has to be minimized for long term viability of such screens. The transfection reagents developed by RJH Biosciences are particularly suitable for library screens. They are provided in homogenous solutions that minimize well-to-well variations during screens. They can be formulated to function in different format, either delivering library members on their own or in combination with other reagents. This application note summarizes experience with screening of two libraries of siRNAs focussing on siRNAs against apoptosis-related and cell cycle proteinsMaterials and Methods:
- Using a robotics workstation, seed cells in 96-well plates in 90 μL medium/well. Incubate for 24 hr.
- Prepare 1.0 μM siRNA dilution plates from 96-well stock libraries with known siRNA concentrations.
- Spot new 96-well plates with desired volume of aliquots from the 1.0 μM dilution plate sets.
- Add desired volume of transfection reagent to siRNA plates. Incubate for 30 min for complex formation. We recommend a total volume of 40 μL to be prepared, where 30 μL will be used below.
- Add 10 μL of complex solution to cells in triplicate (1 well on 3 separate plates) to give 50 nM siRNA.
- Incubate cells at 37°C and assay as needed after a desired period.
- Combination of siRNAs and/or drugs could be added as needed.
Figure 1: Relative growth of MDA-MB-435 cells and skin fibroblasts after treatment with apoptosis-related siRNAs. The growth was normalized against non-treated cells (taken as 100%).
Results and Discussion
In Vivo RNA-Fect reagent was used in two screens. In the first screen, an siRNA library against apoptosis related proteins (Dharmacon siGENOME Human Apoptosis siRNA Library) was employed to identify targets that can specifically inhibit the growth of MDA-MB-435 cells. A total of 446 genes (4 siRNA cocktail/gene) were targeted in this library that resulted in a total of 18 96-well pates to be used for this screen. Human skin fibroblasts were screened with the same library to identify differentially acting siRNAs. As can be seen in Figure 1, the responses of MDA-MB-435 cells to siRNA treatment were generally greater than the fibroblasts. The fibroblasts displayed relatively little decreases in growth as a result of siRNA treatment (<20% inhibition of growth). Numerous siRNAs were readily identified that provided strong inhibition of cell growth (>50%) specifically in MDA-MB-435 cells.In a second screen, a relatively small siRNA library targeting 169 genes involved in cell cycle regulations (Dharmacon siGENOME Cell Cycle Regulation siRNA library; 169 members) was used to assess the effect of siRNAs on the cytotoxicity of two common cancer drugs (paclitaxel and doxorubicin). As can be seen in Figure 2, the results indicated significant variations in the response to siRNA treatment alone: growth inhibition ranged from no effect to ~70% inhibition. For some siRNAs, adding the drug to the treatment did not yield any beneficial effect (e.g., siRNA #97), but the beneficial effects of paclitaxel, more so than the doxorubicin, was evident in some cases (e.g., siRNA #17).
Figure 2:Relative growth of MDA-MB-435 cells after treatment with cell cycle siRNAs with/without paxlitaxel (PXT) and doxorubicin (DOX). The growth was normalized against non-treated cells (taken as 100%).
Benefits of RJH Transfection Reagents:
• High transfection efficiency tailored for specific cell types.• Simple protocol that is amenable for automation and scale-up.
• Minimal toxicity on target cells allowing library effects to be manifested without complication.
• Cost-effective reagent minimizing additional costs in library screens due to transfection reagen.t
• Possibility of using the same transfection reagent in animal models, leading to consistent studies.
References:
• Aliabadi et al., J. Controlled Release (2013) 172: 219-228.• Parmar et al., Frontiers Bioeng. Biotechnol. (2015) 3:14.
• Malo et al., Nat. Biotech. (2006) 24: 167-175.