Confirm Target Gene Knockdown with Fluorescent RNAi Studies

RNAi Analysis Using Near-Infrared Fluorescence

RNAi Analysis Using Western Blots

Quantitative Western blotting is an essential tool for every step in your RNAi studies. With quantitative Western blots, you can:

  • Choose an RNAi vehicle (siRNA or shRNA or other RNA-producing vector construct)
  • Confirm level of knockdown of the target gene
  • Determine the impact of target gene knockdown on a phenotype such as cell proliferation, cell migration, cell cycle, or cell signaling pathways

The Odyssey® M and Odyssey DLx Infrared Imagers provide 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.
figure 1
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
Figure 2. Clear, detailed blot images of 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.

RNAi Analysis Using In-Cell Western™ Assays

The In-Cell Western Assay can be used in a functional siRNA screen measuring the effects of knockdowns in cultured cells.

In-Cell Western assays offer additional throughput for more complex studies, as well as exceptionally consistent data (Z'-factor).1 The ICW has been demonstrated as a powerful cellular assay for genome-wide RNAi screens.2

Hoffmann et al.2 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.
  • A pilot small molecule screen was performed with a library of ~2500 compounds.
  • 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, several 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 replicability.

figure 3
Figure 3. Schematic of the workflow for the siRNA screen. Adapted from Greg Hoffman, Harvard.
figure 4
Figure 4. (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 consistency is shown for a representative plate from the small molecule library.
figure 5
Figure 5. Details of experimental conditions comparing In-Cell Western assays with high-content microscopy.

Resources

References

  1. Boveia, V et al. (2009). Using the Z’-Factor Coefficient to Monitor Quality of Near-Infrared Fluorescent Cell-Based Assays. LI-COR Biosciences.
  2. Hoffmann et al. (2008). A functional siRNA screen for novel regulators of mTORC1 signaling. Poster presentation, ASCB Annual Meeting.