Selection of an optimal RNA transfection reagent and comparison to electroporation for the delivery of viral RNA

Abstract

The initiation of viral RNA replication by the transfection of viral RNA is an integral tool in dissecting the life cycles, susceptibility, and pathogenesis of numerous RNA viruses. Many different transfection methods deliver viral RNA into mammalian cells, including DEAE-dextran and lipidbased reagents, but electroporation is one of the most popular methods. Unfortunately, electroporation suffers from many limitations, including high cell death, serum-free transfection conditions, and requires many cells and relatively large amounts of RNA. To optimize and facilitate the introduction of viral RNAs into mammalian cells, different commercially available RNA transfection reagents were compared for their ability to deliver yellow fever virus (YFV) and hepatitis C virus (HCV) RNA replicons into Huh7 cells. The performance of the commercial transfection reagents was also compared directly to electroporation. When properly optimized, certain reagents were superior to electroporation, with much less cell death, less RNA required and increased transfection efficiency. The factors associated with high efficiency transfection, and the advantage of being able to deliver RNA in the presence of serum are discussed.

Augmentation of Epithelial Resistance to Invading Bacteria by Using mRNA Transfections

“…Apoptosis in untransfected and transfected cells was indistinguishable. Our observations are consistent with previous reports using many different cell types and mRNA packaging systems (35,36). To minimize the loss of cell viability, the delivery system may need to be modified to reduce epithelial cytotoxicity.…”

Optimization of transfection methods for Huh-7 and Vero cells: A comparative study

“…Although Huh-7 and Vero cell lines have been employed in number of prior studies and were transfected via different transfection reagents or electroporation in separate set of experiments but, none of the previous reports provided an optimized transfection protocol for these important cell types based on a comparative study [18][19][20][21][22]. This is specially important for Huh-7 which is generally known as a difficult to transfect cell line [23] and when high rates of transfection efficiencies are determinant for the aim of the study. Previously the elegant study of Uchida and co-workers [24] based on a comparative analysis for the transfection efficiency and cytotoxicity of six non-viral chemical reagents (Lipofectin, Lipofectamine plus, SuperFect, Effectene, DMRIE-C and DOTAP) intended for a wide range of human cells including Huh-7 was reported.…”

A Protocol for Analyzing Hepatitis C Virus Replication

“…High quality RNA is recommended for a higher viral titer yield. For transfecting HCV genomic RNA, polymer and lipid based transfection agents can also be used 21,22 . Viral and host factors greatly influence the growth kinetics of HCV strains.…”