Work flow for multiplexing siRNA assays by solid-phase reverse transfection in multiwell plates
Work flow for multiplexing siRNA assays by solid-phase reverse transfection in multiwell plates
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DOI：
10.1177/1087057108320133
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发表时间：
2008-08-01
影响因子：
--
通讯作者：
Pepperkok, Rainer
中科院分区：
文献类型：
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作者：
Erfle, Holger;Neumann, Beate;Pepperkok, Rainer
细胞芯片固相反向转染技术是一种高通量的哺乳动物细胞平行转染方法。然而，迄今为止，以这种方式转染的细胞仅限于基于显微镜的分析。分析方法，如逆转录酶-聚合酶链反应（RT-PCR）和获得更高的细胞微阵列。我们已经开发了一种快速可靠的协议，用于在多孔板中用siRNA自动化固相反向转染人细胞，以补充细胞微阵列上的固相反向转染。该方法保留了固相反向转染的所有优点，如制造后的长期储存能力，细胞毒性降低，以及与多孔板中的液相转染相比，每次筛选的成本降低。该方案已在不同细胞系（包括U20 S、RPE 1、A549和HeLa细胞）中测试了RNAi介导的几种基因敲低。我们知道，即使在“准备好转染”多孔板生产3个月后，通过RT-PCR和荧光显微镜核表型分析评估的转染效率也没有降低。我们的结论是，在多孔板中的固相反向转染是一种具有成本效益和灵活的工具，用于多重细胞测定。
Solid-phase reverse trasfection on cell microarray 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 microarrays. We have developed a quick and reliable protocol for automated solid-phase reverse trasfection of human cells with siRNAs in multiwell plates complementing solid-phase reverse transfection on cell microarrays. The methos 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 ahow 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.