Use of microarrays in transfection of mammalian cells with dicer-digested small interfering RNAs.
Transfection cell arrays provide promising methods for the high-throughput analysis of gene functions. Such analysis is performed efficiently by using small interfering RNA (siRNA). To extend the usefulness of microarrays, this study was devoted to implementing siRNA prepared from complementary DNA (cDNA). The preparation of siRNA involves the transcription of cDNA to generate double-stranded RNA (dsRNA) followed by digestion with endoribonuclease, dicer, by which one can obtain the library of siRNAs without target sequence optimization. In this study, endoribonuclease-digested siRNA (d-siRNA) was prepared using cDNA encoding enhanced green fluorescent protein (EGFP) and loaded onto micropatterned substrates through electrostatic interactions of siRNA complex with substrates. We observed that d-siRNA loading was comparable to the case with siRNA prepared by chemical synthesis. When cotransfected with EGFP plasmid into human embryonic kidney 293 (HEK293) cells on a microarray, d-siRNA suppressed EGFP expression in a loading-dependent manner. In addition, d-siRNA triggered gene silencing at a level similar to that of chemically synthesized siRNA. The similarity between the two types of siRNA regarding silencing efficiency suggests that heterogeneity in nucleotide sequences of d-siRNA has minor effects. From these results, we conclude that the combination of d-siRNA with the array technology provides useful tools for high-throughput screening of gene functions.
Fujimoto
Hiroyuki
H
Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.
Kato
Koichi
K
Iwata
Hiroo
H
D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D043244:Ribonuclease III / Q000378:metabolism*; D013997:Time Factors; D046888:Tissue Array Analysis / Q000379:methods*; D014162:Transfection / Q000379:methods*
D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D043244:Ribonuclease III