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 CLx 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

^1. Boveia, V et al. Using the Z’-Factor Coefficient to Monitor Quality of Near-Infrared Fluorescent 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.

• 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, 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.

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)

Publishing with LI-COR data?

PubMed

Odyssey Publications: RNA interference 1/2007-6/2009

• Westerns and RNAi

  • Agromayor, M et al.
    Essential role of hIST1 in cytokinesis.
    Mol Biol Cell. 20(5):1374-87 (2009)
    Agromayor et al. examined the effect of hIST1 (sodium tolerance 1) knockdown by siRNA on the protein levels of EGFR. Using Odyssey Quantitative Western blots, they concluded that hIST depletion had no effect on EGFR degradation over a time course of EGF stimulation.

  • Ghosh, P et al.
    Activation of Gαi3 triggers cell migration via regulation of GIV.
    J Cell Biol. 182(2):381-93 (2008)
    In the study of cell migration in response to chemotactic stimuli, Ghosh et al. used Odyssey Quantitative Western blots to assess the impact of G?i3 knockdown on Akt activation. In G?i3 -silenced cells, the peak activation (pAkt/Total Akt) was reduced by 60%. They were also able to demonstrate the quantitative rescue of Akt activation.

  • Funasaka, T et al.
    Down-regulation of phosphoglucose isomerase/autocrine motility factor results in mesenchymal-to-epithelial transition of human lung fibrosarcoma cells.
    Cancer Res. 67(9): 4236-43 (2007)
    Funasaka et al. examined impact of an siRNA for PGI on the expression of Snail and Slug transcription factors with by RT-PCR and Odyssey Quantitative Western blot analysis. The quantitative results were very similar.

  • Shuomin, Z et al.
    MicroRNA-21 targets the tumor suppressor gene tropomyosin 1 (TPM1).
    J Biol Chem. 282(19): 14328-36 (2007)
    Zhu et al. studied the regulation of TPM1 (tropomyosin 1) tumor suppressor by micro RNA-21 by studying a construct where the 3’UTR binding site for mir-21 was deleted. They observed a reduction in TPM1 protein levels by quantitative Western blot on Odyssey, but no effect on the TPM1 mRNA level was detected by real-time qRT-PCR). These results suggested that the mir-21 binding site present in the TPM1-UTR region is critical for mir-21-mediated regulation at the translational level.

  • Liu, Z et al.
    The rho-specific guanine nucleotide exchange factor Dbs regulates breast cancer cell migration.
    J Biol Chem. 284(23):15771-80 (2009)

  • Ha, CH et al.
    A novel role of vascular endothelial cadherin in modulating c-Src activation and downstream signaling of vascular endothelial growth factor.
    J Biol Chem. 283(11):7261-70 (2008)

  • Lehtonen, S et al.
    The endocytic adaptor protein ARH associates with motor and centrosomal proteins and is involved in centrosome assembly and cytokinesis.
    Mol Biol Cell. 19(7):2949-61 (2008)

• Reverse Protein Arrays and RNAi

  • Sahin, O et al.
    Combinatorial RNAi for quantitative protein network analysis. Proc Natl Acad Sci U S A. 104(16):6579-84 (2007)

• In-Cell Western™ and RNAi

  • 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.

Related Products

Odyssey® CLx Infrared Imaging System In-Cell Western™ Kits and Stains Blocking Buffers

IRDye® Secondary Antibody Conjugates IRDye® Streptavidin Conjugates IRDye® Protein Labeling Kits