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

Cell Biology/Biophysics Unit, EMBL, Heidelberg, Germany.
Journal of Biomolecular Screening (Impact Factor: 2.42). 08/2008; 13(7):575-80. DOI: 10.1177/1087057108320133
Source: PubMed


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.

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Available from: Beate Neumann, Apr 13, 2015
    • "RNAi siRNA - spotted microarrays were generated as previously described ( Erfle et al . , 2008 ) in a 1 - well LabTEK ( Thermo Fisher Scientific ) . In brief , the transfection mix was prepared by combining 0 . 4 M sucrose / Opti - MEM ( Life Technologies ) , Lipofectamine 2000 ( Life Technol - ogies ) diluted 1 : 2 in ddH 2 0 , and 3 µM siRNA at 1 . 7 : 1 : 2 . 8 ratio and incubated for 20 min at room temperature in a 384 - well"
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    • "The siRNA-gelatin transfection solution was prepared in 96-well plates (NalgeNunc, Rochester, NY) as described before [12], using a Microlab STAR (Hamilton, Reno, NV) liquid handling robot with the following modifications: 5 μL of siRNA solution (400 ng/μL), 3 μL Opti-MEM (Invitrogen , Darmstadt, Germany) containing 0.4 M sucrose and 3.5 μL Lipofectamine 2000 (Invitrogen) were mixed and incubated for 30 minutes at room temperature. After incubation, 7.25 μL of gelatin/fibronectin mix (1 μL fibronectine to 100 μL gelanine, both Sigma-Aldrich (Taufkirchen, Germany) was added. "
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