Frontiers in RNAi

RNA interference (RNAi) is a biological process in which small nucleotide sequences destroy specific mRNA molecules and, therefore, interrupt genetic expression. RNAi has rapidly developed into a ...
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A Three-Dimensional Spheroid Cell Culture Model for Robust High-Throughput RNA Interference Screens

Pp. 215-231 (17)

Geoffrey Bartholomeusz and Arvind Rao


The sequencing of the human genome and the discovery that synthetic siRNA between 19mer and 22mer could silence genes led to the development of siRNA libraries capable of targeting all known genes within a genome. The emergence of high throughput genetic screens represent a powerful unbiased approach for identifying new targets and may fundamentally change biological research by increasing the speed with which disease mechanisms and potential drug targets can be identified. High-throughput RNAi screens are typically performed using two-dimensional monolayer cell culture models due to ease, convenience, and high cell viability. Although conventional two dimensional cell culture systems have improved our understanding of basic cell biology, the morphology and physiology of cells grown as monolayers in dish cultures differ substantially from the morphology and physiology of cells grown in vivo within a complex three-dimensional microenvironment. There is now a growing realization that 3D cell culture models are superior in biological studies. Three dimensional cell culture models can boost the physiological relevance of cell-based assays and advance the quantitative modeling of biological systems, from cells to organisms. These models exhibit a high degree of structural complexity and homeostasis, analogous to the complexity and homeostasis of tissues and organs. In this chapter we discuss 3D cell culture models and describe a three dimensional spheroid cell culture system and the standard operating procedure for its successful use in high throughput RNAi screens.


3D cell culture, high content image analysis, high Throughput screening, matrix-free nanoculture plates, RNAi, spheroid cell culture.


Department of Experimental Therapeutics, Division of Cancer Medicine, The University of Texas M.D.Anderson Cancer Center, Houston, TX.77054, USA.