Choice of the adequate detection time for the accurate evaluation of the efficiency of siRNA-induced gene silencing.
RNA interference (RNAi) mediated by small interfering RNA (siRNA) has become a popular tool of examining the function of various genes. However, many studies have failed to identify any inhibitory effect of the siRNAs on the expression of the target gene, even though the siRNA being tested had been designed sequence-specifically. In order to determine if this failure is due to the incorrect choice of observation time rather than that of the target site of the gene of interest, this study examined the RNAi efficiency of a vector-driven siRNA targeting two different reporter proteins, EGFP and d2EGFP, whose targeted sequences were identical but the half-lives within the cells differed remarkably from each other (>24h versus 2h), during the time course after transfection. The EGFP expression levels in both cells were reduced in time-dependent manner but the reduction patterns were quite different from each other. The RNAi efficiency varied among the different observation time points and the time required for the maximum RNAi efficiency was proportional to the half-life of the target protein. Stable knocked down cell lines for EGFP expression were then established and the reduced EGFP expression levels in these cell lines were retained for a long period. These results suggest that the choice of an adequate observation time or the establishment of stable knocked down cells by antibiotic selection might be required for making an accurate evaluation of the RNAi effect on the target protein possessing a long half-life.
Choi
Inho
I
Laboratory of Immunology, Transplantation Research Institute and Clinical Research Institute, Seoul National University Hospital, Seoul 110-744, Republic of Korea.
Cho
Bum-Rae
BR
Kim
Donghee
D
Miyagawa
Shuji
S
Kubo
Tomoko
T
Kim
Jae Young
JY
Park
Chung-Gyu
CG
Hwang
Woo Suk
WS
Lee
Jung Sang
JS
Ahn
Curie
C
D000818:Animals; D019556:COS Cells; D018929:Cell Culture Techniques; D002460:Cell Line; D002522:Chlorocebus aethiops; D002999:Clone Cells; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation / Q000187:drug effects; D020868:Gene Silencing; D018390:Gene Targeting; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D007211:Indoles; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D005456:Fluorescent Dyes; D007211:Indoles; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C007293:DAPI; D008156:Luciferases