Berardo Clarissa, Siciliano Veronica, Di Pasqua Laura G, Richelmi Plinio, Vairetti Mariapia, Ferrigno Andrea
Department of Internal Medicine and Therapeutics, University of Pavia.
Eur J Histochem. 2019 Aug 6;63(3):3048. doi: 10.4081/ejh.2019.3048.
RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-transfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have higher viability with respect to Lipofectamine RNAiMAX-administered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.
RNA干扰是一种用于治疗和实验目的以了解基因功能的强大方法。由于对各种肝细胞系基因沉默的了解不足，这项工作旨在比较两种转染试剂，基于脂质体的Lipofectamine™ RNAiMAX和基于聚合物的HepG2特异性GenMute™，在两种人肝癌细胞模型HepG2和Huh7.5中进行比较。在第一部分中，我们通过细胞成像分析评估了荧光Cy3标记的阴性对照siRNA的转染效率；我们发现，与用Lipofectamine RNAiMAX转染的细胞相比，用GenMute处理的细胞在HepG2和Huh7.5细胞中对荧光阴性对照siRNA的摄取更高。在第二部分中，我们通过RT-PCR评估了两种转染试剂对GAPDH的沉默效果，观察每次转染处理后相似的GAPDH mRNA表达。最后，我们通过MTT法测量细胞活力，观察到用GenMute转染的细胞相对于用Lipofectamine RNAiMAX处理的细胞具有更高的活力。这些结果表明，GenMute试剂可能被认为是用于肝脏基因沉默的最合适的转染试剂。
