Erfle, H. et al. Reverse transfection on cell arrays for high content screening microscopy. Nature Protoc. 2, 392-399

MitoCheck Project Group, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Nature Protocol (Impact Factor: 9.67). 02/2007; 2(2):392-9. DOI: 10.1038/nprot.2006.483
Source: PubMed


Here, we describe a robust protocol for the reverse transfection of cells on small interfering (siRNA) arrays, which, in combination with multi-channel immunofluorescence or time-lapse microscopy, is suitable for genome-wide RNA interference (RNAi) screens in intact human cells. The automatic production of 48 'transfection ready' siRNA arrays, each containing 384 samples, takes in total 7 h. Pre-fabricated siRNA arrays can be used without loss of transfection efficiency at least up to 15 months after printing. Different human cell lines that have been successfully transfected using the protocol are presented here. The present protocol has been applied to two genome-wide siRNA screens addressing mitosis and constitutive protein secretion.

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    • "(F) nucleus staining (G) overlay images. for high content screening microscopy has been published in 2007 [8]. Automated Genome wide screening of the NF-kappa-B pathway from genome to single cell using cellular microarrays is shown in Figure 1. "

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    • "FMP/Amiloride Assay The live-cell assay used to identify novel regulators of ENaC activity was performed with the voltage-sensitive dye FMP and 30 mM Amil, as described (Almaç a et al., 2011) on A549 cells reverse-transfected for 48 hr with different siRNAs (Erfle et al., 2007). "
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    ABSTRACT: Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators. Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of (diacylglycerol kinase, iota) DGKι, a protein involved in PiP2 metabolism, downgrades ENaC activity, leading to normalization of both Na(+) and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients.
    Cell 09/2013; 154(6):1390-400. DOI:10.1016/j.cell.2013.08.045 · 32.24 Impact Factor
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    • "Hence, reverse transfection formulations on a surface formed by lyophilization may provide an easy and efficient transfection approach for miRNA delivery. To our knowledge, although DNA and siRNAs have been plated on tissue culture plates for reverse transfection, there has been no such attempt using miRNAs.21,28,29 "
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    ABSTRACT: MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 μL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and -20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall, the miRNA reverse transfection formulation developed in this study is a promising approach for miRNA transfection which can control stem cell fate and is suitable for loading miRNAs onto various biomaterials.
    International Journal of Nanomedicine 05/2013; 8:1595-607. DOI:10.2147/IJN.S43244 · 4.38 Impact Factor
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