The apparent uptake of fluorescently labeled siRNAs by electroporated cells depends on the fluorochrome.
Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after electroporation. We show that Cy3 and Cy5-labeled siRNAs cause a significant amount of cell fluorescence, as judged by flow cytometry. In contrast, several fluorescein-labeled siRNAs could not be detected. Nevertheless, such fluoresceinated siRNAs efficiently suppressed a leukemic target gene, demonstrating that siRNA uptake must have taken place. Therefore, for cell electroporation, fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake.
Cancer Research U.K. Medical Oncology Laboratory, Medical College of St. Bartholomew's Hospital, London, U.K.
Jenny
J
Dunne
Bettina
B
Drescher
Heidemarie
H
Riehle
Philipp
P
Hadwiger
Bryan D
BD
Young
Jürgen
J
Krauter
Olaf
O
Heidenreich
D001483:Base Sequence; D001692:Biological Transport; D002232:Carbocyanines / Q000493:pharmacokinetics; D045744:Cell Line, Tumor; D018274:Electroporation / Q000379:methods; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia; D034741:RNA, Small Interfering / Q000493:pharmacokinetics*; D013379:Substrate Specificity; D020298:U937 Cells
D002232:Carbocyanines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D034741:RNA, Small Interfering; C085321:cyanine dye 5