Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017;7(15):e2435.
doi: 10.21769/BioProtoc.2435. Epub 2017 Aug 5.

RNA Interference Screening to Identify Proliferation Determinants in Breast Cancer Cells

Yong-Wei Zhang  1 , Rochelle E Nasto  1   2   3 , Sandra A Jablonski  1 , Ilya G Serebriiskii  2   4 , Rishi Surana  1 , Joseph Murray  1 , Michael Johnson  1 , Rebecca B Riggins  1 , Robert Clarke  1 , Erica A Golemis  2 , Louis M Weiner  1
Affiliations

RNA Interference Screening to Identify Proliferation Determinants in Breast Cancer Cells

Yong-Wei Zhang et al. Bio Protoc. 2017.

Abstract

RNAi screening technology has revealed unknown determinants of various biological signaling pathways in biomedical studies. This protocol provided detailed information about how to use RNAi screening to identify proliferation determinants in breast tumor cells. siRNA-based libraries targeting against Estrogen receptor (ER)-network, including 631 genes relevant to estrogen signaling, was constructed for screening in breast cancer cells. Briefly, reverse transfection of siRNA induced transient gene knockdown in MCF7 cells. First, the transfection reagent for MCF7 cells was selected. Next, the Z'-score assay was used to monitor if screening conditions yielded efficiently. Then, the ER-network siRNA library screening was preceded by automatic machines under optimized experimental conditions.

Keywords: Breast cancer; CyBio automatic dispenser; Estrogen receptor (ER); Multidrop Combi-nL reagent dispenser; RNA interference (RNAi); Screening; WellMate microplate dispenser; Z′-score.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.. Layout of transfection reagent selection plate.
Efficiency of a variety of transfection reagents was tested in MCF7 cells.
Figure 2.
Figure 2.. Layout of siRNA Z’-score plate
Figure 3.
Figure 3.. Cybio program to dispense siRNA into experimental plates.
A. The first part of program; B. The second part of program.
Figure 4.
Figure 4.. Representative data of Z’-score test.
A. Viability measurement by Cell Titre Blue reading. Purple wells: siDEATH-transfected cells; white wells: siNEG-transfected cells. B. Dot plots of viability measurements. Each dot indicated the value of cell tire blue reading in each well. Upper line of dots indicated those wells containing siNEG-transfected cells; lower line of dots indicated those wells with siDEATH-transfected cells.
Figure 5.
Figure 5.. Layout of ER network siRNA library plate #1-10
Figure 6.
Figure 6.. Layout of ER network siRNA library plate #11
Figure 7.
Figure 7.. Cybio program to dispense siRNA into RNAi screening experimental plates.
A. The first part of program; B. The second part of program.
See this image and copyright information in PMC

References

    1. Astsaturov I., Ratushny V., Sukhanova A., Einarson M. B., Bagnyukova T., Zhou Y., Devarajan K., Silverman J. S., Tikhmyanova N., Skobeleva N., Pecherskaya A., Nasto R. E., Sharma C., Jablonski S. A., Serebriiskii I. G., Weiner L. M. and Golemis E. A.(2010). Synthetic lethal screen of an EGFR-centered network to improve targeted therapies. Sci Signal 3(140): ra67. - PMC - PubMed
    1. Birmingham A., Selfors L. M., Forster T., Wrobel D., Kennedy C. J., Shanks E., Santoyo-Lopez J., Dunican D. J., Long A., Kelleher D., Smith Q., Beijersbergen R. L., Ghazal P. and Shamu C. E.(2009). Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods 6(8): 569-575. - PMC - PubMed
    1. Boutros M., Ahringer J.(2008). The art and design of genetic screens: RNA interference. Nat Rev Genet 9(7): 554-566. - PubMed
    1. Murray J.C., Aldeghaither D., Wang S., Nasto R. E., Jablonski S. A., Tang Y., Weiner L. M.(2014). c-Abl modulates tumor cell sensitivity to antibody-dependent cellular cytotoxicity. Cancer Immunol Res 2(12):1186-1198. - PMC - PubMed
    1. Zhang J. H., Chung T. D. and Oldenburg K. R.(1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4(2): 67-73. - PubMed

LinkOut - more resources