利用RefSeeker R包对RT-qPCR数据归一化候选参考基因的表达稳定性分析。

Patrick H D Petersen, Joanna Lopacinska-Jørgensen, Claus K Høgdall, Estrid V Høgdall
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引用次数: 0

摘要

在对编码RNA(如mRNA)或非编码RNA(如miRNA)进行表达分析时,逆转录定量实时聚合酶链反应(RT-qPCR)是一种广泛使用的方法。为了使这些数据规范化,必须确定一个或多个稳定的内生参考。RefFinder是一个基于网络的在线工具,使用四种几乎普遍使用的算法来评估候选的内源性参考文献-delta- ct, BestKeeper, geNorm和Normfinder。然而,在线界面目前是繁琐和耗时的。我们开发了一个R包RefSeeker,它通过支持原始数据导入和计算每个算法的稳定性来执行简单直接的RefFinder分析,并提供数据输出工具来创建图形和表格。该协议使用RefSeeker R包进行快速简单的RefSeeker稳定性分析。使用五种算法进行稳定性分析:Normfinder, geNorm, delta-Ct, BestKeeper和RefFinder。RT-qPCR数据归一化内源对照物的鉴定。创建可发布的图形和表输出。逐步指导对话框窗口的新手R用户。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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Expression Stability Analysis of Candidate References for Normalization of RT-qPCR Data Using RefSeeker R package.

When performing expression analysis either for coding RNA (e.g., mRNA) or non-coding RNA (e.g., miRNA), reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used method. To normalize these data, one or more stable endogenous references must be identified. RefFinder is an online web-based tool using four almost universally used algorithms for assessing candidate endogenous references-delta-Ct, BestKeeper, geNorm, and Normfinder. However, the online interface is presently cumbersome and time consuming. We developed an R package, RefSeeker, which performs easy and straightforward RefFinder analysis by enabling raw data import and calculation of stability from each of the algorithms and provides data output tools to create graphs and tables. This protocol uses RefSeeker R package for fast and simple RefFinder stability analysis. Key features Perform stability analysis using five algorithms: Normfinder, geNorm, delta-Ct, BestKeeper, and RefFinder. Identification of endogenous references for normalization of RT-qPCR data. Create publication-ready graphs and tables output. Step-by-step guide dialog window for novice R users.

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