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颜林林
(2022-06-10 07:29):
#paper doi:10.1186/1471-2199-7-3 BMC Molecular Biology, 2006, The RIN: an RNA integrity number for assigning integrity values to RNA measurements. 在分子生物学实验中，涉及到RNA质控，评估其分子完整性，最重要且最广泛使用的指标，当属RIN值（RNA integrity Number）。16年前的这篇文章，正是关于RIN值算法及建立过程的工作。Agilent、Roche 和 Quantiom bioinformatics 等单位参与了此项工作。该算法成为至今仍在使用的 2100 生物分析仪的标配和重要输出指标。在RIN值之前，通常使用 28S和18S rRNA的比值来进行评估（一般要求达到至少2.0），而这个比值受电泳胶图展示和手工测量的影响，经常不够稳定，在实验室之间存在很大差异，更重要的，其与RNA分子的完整性经常并不相关。于是，本文开发了一套方法，使用毛细管电泳技术，采集到样本中的所有不同长度核酸分子的丰度信息，由此自动提取特征，基于贝叶斯方法和神经网络算法，构建回归模型，并最终选择出估计RNA完整性的特征组合，并计算出RIN值（取值1-10，1代表完全降解，10代表无降解）。研究者从人、大鼠、小鼠的不同器官组织，以及各类细胞系中，分别提取了RNA，共收集了1208份样本，这其中主要是未降解的完整样本和完全降解的样本，此外也包括了足够的部分降解的样本。通过不同比例的组合将其混合，构造了一套包含各种不同降解程度的实际样本，用于产出数据、提取特征后构造分类模型，以及对该分类模型（模型输出为RIN值，分别为1-10的整数）的性能评估。同时也将模型算法计算得到的rRNA比值、RIN值，与（4个看家基因的）rtPCR数据进行了对比，确认其对RNA质量和完整程度的代表性。
Abstract:
BACKGROUND: The integrity of RNA molecules is of paramount importance for experiments that try to reflect the snapshot of gene expression at the moment of RNA extraction. Until recently, there …
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BACKGROUND: The integrity of RNA molecules is of paramount importance for experiments that try to reflect the snapshot of gene expression at the moment of RNA extraction. Until recently, there has been no reliable standard for estimating the integrity of RNA samples and the ratio of 28S:18S ribosomal RNA, the common measure for this purpose, has been shown to be inconsistent. The advent of microcapillary electrophoretic RNA separation provides the basis for an automated high-throughput approach, in order to estimate the integrity of RNA samples in an unambiguous way.METHODS: A method is introduced that automatically selects features from signal measurements and constructs regression models based on a Bayesian learning technique. Feature spaces of different dimensionality are compared in the Bayesian framework, which allows selecting a final feature combination corresponding to models with high posterior probability.RESULTS: This approach is applied to a large collection of electrophoretic RNA measurements recorded with an Agilent 2100 bioanalyzer to extract an algorithm that describes RNA integrity. The resulting algorithm is a user-independent, automated and reliable procedure for standardization of RNA quality control that allows the calculation of an RNA integrity number (RIN).CONCLUSION: Our results show the importance of taking characteristics of several regions of the recorded electropherogram into account in order to get a robust and reliable prediction of RNA integrity, especially if compared to traditional methods.
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