Sodowich Bruce I, Fadl Irini, Burns Craig
Roche Molecular Systems, Branchburg, NJ 08876-3733, USA.
Electrophoresis. 2007 Jul;28(14):2368-78. doi: 10.1002/elps.200600673.
The Agilent 2100 Bioanalyzer can characterize in vitro RNA transcripts for their integrity, purity, concentration, and size. The results are comparable to denatured agarose electrophoresis with ethidium bromide staining and UV spectrophotometry combined. In this report, we describe our strategy for validating this method following the International Conference on Harmonization guidelines. The assay has a linear range of quantitation between 500 and 25 ng/microL. Quantitation accuracy is within +/-20% of measurements produced from spectrophotometry and sizing accuracy is within +/-7% based on theoretical sizes. Concentration and sizing measurements within a single assay produce RSDs that are <10 and <2%, respectively, indicating good precision. The method also maintains a tolerable precision when altering operator, day, and reagent kit lot. The RSD for quantitation is <or=25 and <2% for sizing. The LOQ and LOD are 15.4 and 5.4 ng/microL based on experimentation, producing values similar to those specified by the manufacturer. The Bioanalyzer can differentiate between the RNA transcript and DNA contamination, and protein contamination quenches the RNA transcript signal. The effect of the ionic strength of the buffer on RNA analysis is also examined. Limitations of this method and future applications are discussed.
安捷伦2100生物分析仪能够对体外转录RNA的完整性、纯度、浓度和大小进行表征。其结果与溴化乙锭染色的变性琼脂糖电泳和紫外分光光度法相结合的结果相当。在本报告中，我们描述了按照国际协调会议指南验证该方法的策略。该测定法的定量线性范围为500至25 ng/μL。定量准确度在分光光度法测量值的±20%以内，大小测定准确度基于理论大小在±7%以内。单次测定中的浓度和大小测量产生的相对标准偏差（RSD）分别<10%和<2%，表明精密度良好。当改变操作人员、日期和试剂盒批次时，该方法也能保持可接受的精密度。定量的RSD<或 = 25%，大小测定的RSD<2%。根据实验，定量下限（LOQ）和检测限（LOD）分别为15.4和5.4 ng/μL，产生的值与制造商规定的值相似。生物分析仪能够区分RNA转录本和DNA污染，并且蛋白质污染会淬灭RNA转录本信号。还研究了缓冲液离子强度对RNA分析的影响。讨论了该方法的局限性和未来应用。
