The effectiveness of double-stranded short inhibitory RNAs (siRNAs) may depend on the method of transfection.
RNA interference (RNAi) is a recently described powerful experimental tool that can cause sequence-specific gene silencing, thereby facilitating functional analysis of gene function. Consequently, we became interested in using RNAi to determine the function of aberrantly expressed ErbB3 in the KAS-6/1 human myeloma cell line. Despite the wealth of information available on the use of RNAi, dsRNA target design, and the transfection of dsRNA in vitro, little information is available for transfecting dsRNA into nonadherent cells from any species. In the present study, we report that gene silencing of ErbB3 was not observed in myeloma cells when dsRNA targeting ErbB3 was introduced using conventional transfection agents and protocols that have proved successful for several adherent cell lines. Silencing of ErbB3, however, was observed in T47D cells, an adherent breast carcinoma cell line, using the same transfection methods, indicating that our target sequence was functional for gene silencing of ErbB3. Interestingly, ErbB3 was silenced in myeloma cells when the dsRNA target was introduced by electroporation. Thus, our studies illustrate the striking dependence of dsRNA-mediated gene silencing in some cells on the methods of dsRNA transfection.
Walters
D K
DK
Department of Immunology, Tumor Biology Program, Mayo Graduate and Medical Schools, Mayo Clinic/Foundation, Rochester, MN 55905, USA.
Jelinek
D F
DF
D018274:Electroporation; D005786:Gene Expression Regulation; D006801:Humans; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000008:administration & dosage* / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured
D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering