Use of bicistronic vectors in combination with flow cytometry to screen for effective small interfering RNA target sequences.
The efficacy and specificity of small interfering RNAs (siRNAs) are largely dependent on the siRNA sequence. Since only empirical strategies are currently available for predicting these parameters, simple and accurate methods for evaluating siRNAs are needed. To simplify such experiments, target genes are often tagged with reporters for easier readout. Here, we used a bicistronic vector expressing a target gene and green fluorescent protein (GFP) to create a system in which the effect of an siRNA sequence was reflected in the GFP expression level. Cells were transduced with the bicistronic vector, expression vectors for siRNA and red fluorescent protein (RFP). Flow cytometric analysis of the transduced cells revealed that siRNAs for the target gene silenced GFP from the bicistronic vector, but did not silence GFP transcribed without the target gene sequence. In addition, the mean fluorescence intensities of GFP on RFP-expressing cells correlated well with the target gene mRNA and protein levels. These results suggest that this flow cytometry-based method enables us to quantitatively evaluate the efficacy and specificity of siRNAs. Because of its simplicity and effectiveness, this method will facilitate the screening of effective siRNA target sequences, even in high-throughput applications.
Kamio
Naoka
N
Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Hirai
Hideyo
H
Ashihara
Eishi
E
Tenen
Daniel G
DG
Maekawa
Taira
T
Imanishi
Jiro
J
D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics / Q000302:isolation & purification*; D014161:Transduction, Genetic; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D000097573:Red Fluorescent Protein