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.

Download full-text


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"
    [Show abstract] [Hide abstract]
    ABSTRACT: Targeting of inner nuclear membrane (INM) proteins is essential for nuclear architecture and function, yet its mechanism remains poorly understood. Here, we established a new reporter that allows real-time imaging of membrane protein transport from the ER to the INM using Lamin B receptor and Lap2β as model INM proteins. These reporters allowed us to characterize the kinetics of INM targeting and establish a mathematical model of this process and enabled us to probe its molecular requirements in an RNA interference screen of 96 candidate genes. Modeling of the phenotypes of genes involved in transport of these INM proteins predicted that it critically depended on the number and permeability of nuclear pores and the availability of nuclear binding sites, but was unaffected by depletion of most transport receptors. These predictions were confirmed with targeted validation experiments on the functional requirements of nucleoporins and nuclear lamins. Collectively, our data support a diffusion retention model of INM protein transport in mammalian cells. © 2015 Boni et al.
    The Journal of Cell Biology 06/2015; 209(5):705-720. DOI:10.1083/jcb.201409133 · 9.83 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Chemoresistance in malignant melanoma remains an unresolved clinical issue. In the search for novel molecular targets a live-cell high-content RNAi screen based on gene expression data was performed in cisplatin-sensitive and cisplatin-resistant MeWo melanoma cells, Mel-28 cells and a melanocyte cell line. Cells were exposed to 91 siRNAs and distinct nucleus-derived phenotypes such as cell division, cell death and migration phenotypes were detected by time-lapse microscopy over 60 hours. Using this approach, cisplatin-sensitive and cisplatin-resistant melanoma cells were compared by automated image analysis and visual inspection. In cisplatin-sensitive MeWo melanoma cells, 14 genes were identified that showed distinct phenotype abnormalities after exposure to gene-specific siRNAs. In cisplatin-resistant MeWo cells, five genes were detected. Nine genes were detected whose knock-down led to differential nuclear phenotypes in cisplatin-sensitive and -resistant cells. In Mel-28 cells, nine genes were identified which induced nuclear phenotypes including all eight genes which were identified in cisplatin-resistant MeWo cells. An analogous RNAi screen on melanocytes revealed no detectable phenotype abnormalities after RNAi. Pathway analysis showed in cisplatin-sensitive MeWo cells and Mel-28 cells an enrichment of at least three genes in major mitotic pathways. We hereby show that siRNA screening may help to identify tumor-specific genes leading to phenotype abnormalities. These genes may serve as a potential therapeutic targets in the treatment of melanoma. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    Biotechnology Journal 04/2015; 10(9). DOI:10.1002/biot.201400501 · 3.49 Impact Factor
  • Source
    • "A collection of 460 different commercially available siRNAs (Ambion, Applied Biosystems) targeting 230 genes was reverse-transfected into HIV-permissive HeLaP4 cells (wild-type or overexpressing Ago2) through an established reverse transfection protocol (49). The individual genes were silenced for 36 h before infection with a Gfp-expressing HIV-1 NL4-3 derivative [AGFP (50)]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.
    Nucleic Acids Research 09/2013; 41(21). DOI:10.1093/nar/gkt836 · 9.11 Impact Factor
Show more