生物样本库RNA质量控制的方法学验证

摘要

目的：对现有的RNA样本质量控制的方法进行方法学验证。方法 RNA样本质量控制往往关注浓度、纯度和完整度3个参数，其中前两者可以用光密度（optical density，OD）260和OD260／280来表征，后者可以用RNA完整度（ RNA integrity number ，RIN）值来衡量。由于OD260／280对于RNA纯度的评价尚无明确标准，故仅对RNA的浓度和完整度展开探讨。按照ISO／IEC 17025∶2005的要求，从精密度、准确度和线性3个方面，对RNA定量进行方法学验证。由于缺乏测量RIN值测定的标准品，RNA完整度的方法学验证仅限于精密度的考察。结果选用的微量紫外分光光度法对RNA标准品进行定量，精密度、准确度和线性均达到要求；Agilent 2100的RIN值系统对人Hela细胞来源的总RNA进行了评价，RIN值的精密度也通过了考察。结论微量紫外分光光度法和Agilent 2100的RIN值系统可以作为RNA样本质量控制的方法。%Objective To perform method validation for RNA quality control .Methods There are three quality controls that are performed on isolated RNA:concentration , purity and integ-rity.The optical density ( OD) at 260 nm is used to determine the RNA concentration in a solution . The ratio of the absorbance at 260 nm to 280 nm is used to assess the RNA purity of an RNA prepa-ration.The integrity assessment is based on the RNA integrity number (RIN) system.In this work, we focused on the RNA concentration and integrity .According to the requirement of ISO/IEC 17025∶2005, the key factors of precision , accuracy and linearity testing were presented in assessing the method of RNA quantity .Precision testing on RNA integrity was also preformed .Results In this study , we focused on the validation of RNA quantitation by spectrophotometry , and performed preci-sion, accuracy and linearity assessment .Total RNA from Hela cells was used for precision testing on RNA integrity.All the data were acceptable .Conclusion The method of spectrophotometry and RIN system are qualified for RNA quality control .