AERIUS® IMAGER
Applications for the Aerius Imaging System
RNAi Screens
Western Blot and RNAi
Quantitative Western blotting is an essential tool for every step in your RNAi studies:
Choosing an RNAi vehicle (siRNA or shRNA or other RNA-producing vector construct),
Confirming level of knockdown of the target gene,
Determining the impact of target gene knockdown on a phenotype such as cell proliferation, cell migration, cell cycle, or cell signaling pathways.
The Odyssey Infrared Imaging System provides accurate digital protein data for your RNAi studies.
Raw data can be normalized for increased accuracy using one of the two independent channels (colors) to monitor a control protein.
Stable fluorescent signal is directly proportional to amount of target for consistent data.
Wide linear range allows knockdown levels involving both weak and strong signals to be measured accurately in a single image without the bleed over of signal from adjacent “blow out” bands.
In-Cell Western assays also offer additional throughput for more complex studies, as well as exceptionally consistent data (Z’-factor).1 The ICW has recently been demonstrated as a powerful cellular assay for genome-wide RNAi screens. 2
[ABOVE] Figure 1. Validation of siRNA knockdown in A431 cells by two-color quantitative Western Each siRNA target is detected in green. In red, tubulin was detected as used as a loading control to normalize for sample variation. Graph indicates % of target expression in siRNA-treated cells, relative to controls. In each blot, the marker is at the left, the negative control is in the center, and the siRNA is at the right.
Figure 2. Clear, detailed blot images. siRNA knockdown of ERK2 was performed in A431 cells. ERK2 detection is shown in green (primary antibody binds both ERK isoforms); tubulin loading control is shown in red. In this Western blot, the spatial resolution of ERK isoforms demonstrates that when ERK2 expression is lowered with siRNA treatment, ERK1 expression increases.
1. Boveia, V et al. Using the Z’-Factor Coefficient to Monitor Quality of Near-Infrared Flourescent Cell-Based Assays. LI-COR Biosciences (2009).
2. Hoffmann et al. A functional siRNA screen for novel regulators of mTORC1 signaling. Poster presentation, ASCB Annual Meeting (2008)
In-Cell Western and RNAi
The In-Cell Western™ can be used in a functional siRNA screen measuring the effects of knockdowns in cultured cells.
Hoffmann et al.1 used In-Cell Western screening to assess the effects of knockdowns on mTORC1-dependent phosphorylation of ribosomal protein S6 (rpS6).
HeLa cells were transfected with siRNA and screened for phosphorylation of prS6 at Ser235/236.
NHS ester cell labeling was used for cell number normalization.
The Dharmacon Human Druggable Genome siRNA library gave 7,317 genes from the human genome that are likely targets for pharmacological inhibition.
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A pilot small molecule screen was performed with a library of ~2500 compounds.
[ABOVE] Figure 1. (A) ) Results of pilot small molecule screen performed with a library of ~2500 known bioactive compounds. Compounds with average Z-score < -2 are considered hits and are shown in red. Known inhibitors of mTORC1 signaling found in the library are shown in green. Star-shaped symbols represent compounds with > 4-fold reduction in cell number.
(B) Plate to plate reproducibility is shown for a representative plate from the small molecule library.
Knockdown of components required for both the growth factor and amino acid branches of the mTORC1 signaling network caused reduction in phospho-rpS6.
Known genes involved in growth factor-mediated inputs leading to rpS6 phosphorylation (IRS2, IGF1R, PI3-kinase, PDK1, and S6K2) scored as hits in the screen, validating the approach.
In addition to known pathway components, a number of uncharacterized genes scored as strong hits.
In-Cell Western RNAi screening was found to be faster and less expensive than high content microscopy (IF), and gave similar or better statistical reproducibility.
Figure 2.
Schematic of the workflow for the siRNA screen (adapted from Greg Hoffman, Harvard)
1. Hoffmann et al. A functional siRNA screen for novel regulators of mTORC1 signaling. Poster presentation, ASCB Annual Meeting (2008)
PubMed
Odyssey Publications: RNA interference 1/2007-6/2009
- Hoffman, G et al.
A functional siRNA screen for novel regulators of mTORC1 signaling.
Poster presentation, ASCB Annual Meeting (2008)
Hoffmann et al. utilized the screening to assess the effects on knockdowns in mammalian tissue culture cells on the mTORC1 dependent phosphorylation of rpS6 at Ser235/236. - Perrimon, N and Mathey-Prevot B.
Applications of high-throughput RNA interference screens to problems in cell and developmental biology.
Genetics 175: 7–16 (2007)
This review discusses the use of infrared fluorescent cell-based assays as a strategy for increased-throughput RNAi screening and an alternative to microscopy and high content screening. - Sklan, EH et al.
A Rab-GAP TBC domain protein binds Hepatitis C virus NS5A and mediates viral replication.
J Virol. 81:11096–11105 (2007)
In this study, isolation of a novel cellular binding partner of Hepatitis C virus (HCV) is reported.; To investigate the role of this factor in establishment of HCV infection, it was depleted by siRNA knockdown prior to HCV inoculation of the cells. An antibody to the HCV core protein was then used to detect infection in fixed cells. Knockdown dramatically reduced the ability of HCV to establish infection.
