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

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

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsLorenzo Galluzzi, Nils-Petter Rudqvist
PublisherAcademic Press Inc.
Pages299-322
Number of pages24
ISBN (Print)9780128206676
DOIs
StatePublished - 2020
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume636
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Publisher Copyright:
© 2020 Elsevier Inc.

Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.

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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