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. 2020:636:299-322.
doi: 10.1016/bs.mie.2019.06.002. Epub 2019 Jul 3.

RNA interference screening methods to identify proliferation determinants and mechanisms of resistance to immune attack

Yong-Wei Zhang  1 , Rochelle E Nasto  2 , Sandra A Jablonski  1 , Ilya G Serebriiskii  3 , Rishi Surana  1 , Joseph Murray  1 , Michael Johnson  1 , Rebecca B Riggins  1 , Robert Clarke  1 , Erica A Golemis  4 , Louis M Weiner  5
Affiliations

RNA interference screening methods to identify proliferation determinants and mechanisms of resistance to immune attack

Yong-Wei Zhang et al. Methods Enzymol. 2020.

Abstract

We have used RNA interference (RNAi) screening technology to reveal unknown components of biological signaling pathways including survival mechanisms of estrogen-independent breast cancer cell growth and cancer cell resistance to immune attack. In this chapter, a detailed protocol describing the use of RNAi screening to identify factors important for the proliferation of estrogen-independent MCF7 breast cancer cells will be described. Resistance to therapies that target the estrogen pathway remains a challenge in the treatment of estrogen receptor-positive breast cancer. To address this challenge, small interfering-RNA (siRNA)-based libraries targeting an estrogen receptor (ER)- and aromatase-centered network, including 631 genes relevant to estrogen signaling, was designed and constructed for RNAi screening. This protocol will include the following parts: (1) selection of RNAi transfection reagent for specific cells; (2) optimization of RNAi screening conditions using Z'-factor; (3) procedure of ER-network gene siRNA library screening using automated machines under optimized experimental conditions; and (4) method of analysis for RNAi screening data to identify specific determinants important for cell proliferation. 46 genes were found to be selectively required for the survival of estrogen-independent MCF7-derived cells.

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

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Figures

Figure 1.
Figure 1.
Outline of the protocol.
Figure 2.
Figure 2.
Layout of transfection reagent selection plate.
Figure 3.
Figure 3.
Workflow of high throughput-RNAi screening
Figure 4.
Figure 4.
Layout of siRNA Z’-factor plate
Figure 5.
Figure 5.
Loading of siRNA plates and experimental plates
Figure 6.
Figure 6.
Cybio program to dispense siRNA into experimental plates.
Figure 7.
Figure 7.
Representative data of Z’-factor 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. hour up to 4 h (as described above).
Figure 8.
Figure 8.
Layout of ER network siRNA library plate #1–10
Figure 9.
Figure 9.
Layout of ER network siRNA library plate #11
Figure 10.
Figure 10.
Cybio program to dispense siRNA into RNAi screening experimental plates.
See this image and copyright information in PMC

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