Selection of an optimal RNA transfection reagent and comparison to electroporation for the delivery of viral RNA.
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 lipid-based 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.
Gonzalez
Gladys
G
Department of Medicine , University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA.
Pfannes
Loretta
L
Brazas
Robert
R
Striker
Rob
R
D045744:Cell Line, Tumor; D018274:Electroporation / Q000379:methods; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism; D006801:Humans; D012328:RNA Viruses / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D012093:Replicon / Q000235:genetics; D014162:Transfection / Q000379:methods*; D015005:Yellow fever virus / Q000235:genetics / Q000378:metabolism
D012367:RNA, Viral