Detection of siRNA administered to cells and animals by using a fluorescence intensity distribution analysis polarization system.
Small interfering RNA (siRNA) has excellent pharmacological features and is expected to be used for therapeutic drug development. To this end, however, new RNA technology needs to be established so that extremely small amounts (less than 1 pmol) of siRNA can be detected in organs of experimental animals and in human blood to facilitate pharmacokinetics studies. An important feature is that this new technology is not dependent on radioisotopes and can detect siRNA molecules identical to those used for drug development in preclinical tests with experimental animals or in clinical tests with humans. We report a convenient method that can detect small amounts of siRNA. The method uses high-power confocal microscopic analysis of fluorescence polarization in DNA probes that are bound to one of the strands of siRNA and directly quantitates the copy number of siRNA molecule after extraction from specimens. A pharmacokinetic study to examine the blood retention time of siRNA/cationic liposomes in mice showed that this straightforward method is consistent with the other reverse transcriptase polymerase chain reaction amplification-based method. We believe that the entire process is simple and applicable for a high-throughput analysis, which provides excellent technical support for fundamental research on RNA interference and development of siRNA drugs.
Abe
Takashi
T
Micro-imaging Systems Division, Olympus Corporation, Tokyo, Japan.
Goda
Kazuhito
K
Futami
Kazunobu
K
Furuichi
Yasuhiro
Y
D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D015342:DNA Probes / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D007275:Injections, Intravenous; D051379:Mice; D018613:Microscopy, Confocal; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000032:analysis* / Q000097:blood; D014018:Tissue Distribution; D014162:Transfection
D015342:DNA Probes; D034741:RNA, Small Interfering