Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia, Athens, GA 30602-2352, United States.
Anal Chim Acta. 2013 Oct 17;799:57-67. doi: 10.1016/j.aca.2013.08.040. Epub 2013 Aug 31.
Bioanalysis of siRNAs is challenging due to their size (5-14 kDa) and negative charge across the backbone, which complicates both sample preparation and chromatography. We present here a one step sample preparation combined with non-denaturing anion exchange chromatography with UV detection for the quantitation of siRNA and its chain shortened metabolites. The sample preparation uses a novel lysis buffer with proteinase K to effectively isolate siRNA from cells and formulated media with greater than 95% recovery. The ion exchange chromatography allows for a lower limit of quantitation of 6 ng mL(-1) in cells and media equivalent to 6 ng/200,000 cells. This method is applied to study the uptake of siRNA in prostate cancer cells and the disappearance in the media and siRNA metabolism. siRNA metabolites are identified by matching the retention time of standards to metabolite peaks. Identification is further confirmed by mass spectrometry. To our knowledge this is the first ion exchange method reported for the quantitation of siRNA from a biological matrix. It is also the first non-denaturing chromatographic method reported for siRNA quantitation.
由于 siRNA 的大小（5-14 kDa）和骨架上的负电荷，其生物分析具有挑战性，这使得样品制备和色谱分离都变得复杂。我们在此介绍一种一步法样品制备，结合非变性阴离子交换色谱和紫外检测，用于定量 siRNA 及其链缩短代谢物。该样品制备使用一种新型裂解缓冲液，其中含有蛋白酶 K，可有效从细胞和配方培养基中分离 siRNA，回收率大于 95%。离子交换色谱的定量下限为细胞和培养基中 6ng mL(-1)，相当于 6ng/200,000 个细胞。该方法用于研究前列腺癌细胞中 siRNA 的摄取以及在培养基中的消失和 siRNA 代谢情况。通过将标准品的保留时间与代谢物峰匹配来鉴定 siRNA 代谢物。通过质谱进一步确认鉴定。据我们所知，这是首次报道用于从生物基质中定量 siRNA 的离子交换方法。这也是首次报道用于 siRNA 定量的非变性色谱方法。
