Abe Takashi, Goda Kazuhito, Futami Kazunobu, Furuichi Yasuhiro
Micro-imaging Systems Division, Olympus Corporation, Tokyo, Japan.
Nucleic Acids Res. 2009 Apr;37(7):e56. doi: 10.1093/nar/gkp131. Epub 2009 Mar 12.
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.
小干扰RNA（siRNA）具有出色的药理学特性，有望用于治疗药物开发。然而，为此需要建立新的RNA技术，以便能够在实验动物的器官和人体血液中检测到极少量（小于1皮摩尔）的siRNA，以促进药代动力学研究。一个重要特点是，这项新技术不依赖于放射性同位素，并且能够检测与在实验动物的临床前试验或人体临床试验中用于药物开发的siRNA分子相同的siRNA分子。我们报告了一种能够检测少量siRNA的便捷方法。该方法利用对与siRNA其中一条链结合的DNA探针中的荧光偏振进行高功率共聚焦显微镜分析，并在从标本中提取后直接定量siRNA分子的拷贝数。一项检测siRNA/阳离子脂质体在小鼠体内血液保留时间的药代动力学研究表明，这种直接的方法与其他基于逆转录聚合酶链反应扩增的方法一致。我们认为整个过程简单且适用于高通量分析，这为RNA干扰的基础研究和siRNA药物开发提供了出色的技术支持。
