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 language||English (US)|
|Title of host publication||Methods in Enzymology|
|Editors||Lorenzo Galluzzi, Nils-Petter Rudqvist|
|Publisher||Academic Press Inc.|
|Number of pages||24|
|State||Published - 2020|
|Name||Methods in Enzymology|
Bibliographical notePublisher Copyright:
© 2020 Elsevier Inc.
Copyright 2020 Elsevier B.V., All rights reserved.
- Breast cancer
- CyBio automatic dispenser
- Estrogen receptor (ER)
- Multidrop Combi-nL reagent dispenser
- RNA interference (RNAi)
- WellMate microplate dispenser