Automated high-throughput siRNA transfection in raw 264.7 macrophages: a case study for optimization procedure.

Jean-Philippe Carralot; Tae-Kyu Kim; Boris Lenseigne; Annette S Boese; Peter Sommer; Auguste Genovesio; Priscille Brodin

Automated high-throughput siRNA transfection in raw 264.7 macrophages: a case study for optimization procedure.

Jean-Philippe Carralot ^{1, *} Tae-Kyu Kim ¹ Boris Lenseigne ¹ Annette S Boese ¹ Peter Sommer ¹ Auguste Genovesio ¹ Priscille Brodin ¹

* Corresponding author

1 Institut Pasteur de Corée

Abstract : RNAi using siRNA is a very powerful tool for functional genomics to identify new drug targets and biological pathways. Although their use in epithelial cells is relatively easy and straightforward, transfection in other cell types is still challenging. The authors report the optimization of transfection conditions for Raw 267.4 macrophage cells. The herein described procedure makes use of automated confocal microscopy, enhanced green fluorescent protein (EGFP)-expressing macrophages, and fluorescently labeled siRNAs to simultaneously quantify both siRNA uptake and silencing efficiency. A comparison of 10 commercial transfectants was performed, leading to the selection of the transfectant giving the highest reproducible knock-down effect without inducing cell toxicity or cell activation. Several buffers used for siRNA/lipid complex assembly were tested, and such a study revealed the crucial importance of this parameter. In addition, a kinetics study led to the determination of the optimal siRNA concentration and the best time window for the assay. In an original approach aimed at simultaneously optimizing both the high-throughput screening process and biological factors, optimal reagent volumes and a process flowchart were defined to ensure robust silencing efficiencies during screening. Such an account should pave the way for future genome-wide RNAi research in macrophages and present an optimization procedure for other "hard-totransfect" cell lines.

Document type :

Journal articles

Domain :

Life Sciences [q-bio] / Microbiology and Parasitology / Virology

Complete list of metadatas

Cited literature [30 references] Display Hide Download

https://hal-riip.archives-ouvertes.fr/pasteur-00683854
Contributor : Peter Sommer <>
Submitted on : Friday, March 30, 2012 - 6:01:11 AM
Last modification on : Monday, October 8, 2018 - 5:44:04 PM
Long-term archiving on : Wednesday, December 14, 2016 - 7:18:51 PM

Carralot_et_al._2009.pdf

Files produced by the author(s)

Automated high-throughput siRNA transfection in raw 264.7 macrophages: a case study for optimization procedure.

File

Identifiers

Collections

Citation

Export

Metrics

470

1006

Contact

Automated high-throughput siRNA transfection in raw 264.7 macrophages: a case study for optimization procedure.

File

Identifiers

Collections

Citation

Export

Share

Metrics

470

1006

Contact