Work Flow for Multiplexing siRNA Assays by Solid-Phase Reverse Transfection in Multiwell Plates

Solid-phase reverse transfection on cell microarrays is a high-throughput method for the parallel transfection of mammalian cells. However, the cells transfected in this way have been restricted so far to microscopy-based analyses. Analysis methods such as reverse transcriptase—polymerase chain reaction (RT-PCR) and access to higher cell numbers for statistical reasons in microscopy-based assays are not possible with solid-phase reverse transfection on cell microarrays. We have developed a quick and reliable protocol for automated solid-phase reverse transfection of human cells with siRNAs in multiwell plates complementing solid-phase reverse transfection on cell microarrays. The method retains all advantages of solid-phase reverse transfection such as long-term storage capacity after fabrication, reduced cytotoxicity, and reduced cost per screen compared with liquid-phase transfection in multiwell plates. The protocol has been tested for the RNAi-mediated knockdown of several genes in different cell lines including U20S, RPE1, A549, and HeLa cells. We show that even 3 months after production of the “ready to transfect” multiwell plates, there is no reduction in their transfection efficiency as assessed by RT-PCR and nuclear phenotyping by fluorescence microscopy. We conclude that solid-phase reverse transfection in multiwell plates is a cost-efficient and flexible tool for multiplexing cellular assays. (Journal of Biomolecular Screening. 2008:575-580)

[1]  Holger Erfle,et al.  siRNA cell arrays for high-content screening microscopy. , 2004, BioTechniques.

[2]  Vivek Mittal,et al.  High-throughput selection of effective RNAi probes for gene silencing. , 2003, Genome research.

[3]  H. Erfle,et al.  High-throughput RNAi screening by time-lapse imaging of live human cells , 2006, Nature Methods.

[4]  D. Sabatini,et al.  Microarrays of cells expressing defined cDNAs , 2001, Nature.

[5]  E. Bossy‐Wetzel,et al.  Recruitment of MKLP1 to the spindle midzone/midbody by INCENP is essential for midbody formation and completion of cytokinesis in human cells. , 2005, The Biochemical journal.

[6]  Robert Lucito,et al.  RNA interference microarrays: high-throughput loss-of-function genetics in mammalian cells. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[7]  R. Pepperkok,et al.  Chromosome-induced microtubule assembly mediated by TPX2 is required for spindle formation in HeLa cells , 2002, Nature Cell Biology.

[8]  Yoann Roupioz,et al.  Quantitative analysis of highly parallel transfection in cell microarrays. , 2004, Nucleic acids research.

[9]  E. Dougherty,et al.  RNAi microarray analysis in cultured mammalian cells. , 2003, Genome research.

[10]  H. Erfle,et al.  Reverse transfection on cell arrays for high content screening microscopy , 2007, Nature Protocols.

[11]  Susan L. Kline,et al.  Deciphering protein function during mitosis in PtK cells using RNAi , 2006, BMC Cell Biology.

[12]  Jan Ellenberg,et al.  Roles of Polo-like Kinase 1 in the Assembly of Functional Mitotic Spindles , 2004, Current Biology.