Abstract
The lack of specific pharmacological tools to interrogate the functional role of palmitoyl acyltransferases (zDHHCs) in mammalian cells has significantly hampered the understanding of this important gene family. Gene silencing by RNA interference (RNAi) is a process in eukaryotes that allows specific knockdown of the expression of proteins by targeting their coding mRNA. RNAi can thus be used as a proteomic tool to study the functional role of specific zDHHCs in cells by analyzing the effects of endogenous zDHHC knockdown on their protein targets or pathways. Here we describe the application of short interfering RNA (siRNA), a class of short (20–25 base pairs) double-stranded RNAs, to knockdown endogenous zDHHC enzymes expressed in human embryonic kidney (HEK293) cells and subsequent validation of knockdown efficiency using RT-qPCR to quantify zDHHC mRNA levels.
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Acknowledgments
This work is dedicated to the memory of our colleague Lijun Tian, who developed and optimized the siRNA approach in the laboratory, and died in service before the writing of this chapter. The work was supported by the Wellcome Trust, British Heart Foundation, and Diabetes UK.
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McClafferty, H., Shipston, M.J. (2019). siRNA Knockdown of Mammalian zDHHCs and Validation of mRNA Expression by RT-qPCR. In: Linder, M. (eds) Protein Lipidation. Methods in Molecular Biology, vol 2009. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9532-5_12
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DOI: https://doi.org/10.1007/978-1-4939-9532-5_12
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