The Renilla luciferase gene as a reference gene for normalization of gene expression in transiently transfected cells.
The Renilla luciferase gene as a reference gene for normalization of gene expression in transiently transfected cells.
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DOI：
10.1186/1471-2199-11-103
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发表时间：
2010-12-31
影响因子：
--
通讯作者：
Pitt AR
中科院分区：
文献类型：
--
作者：
Jiwaji M;Daly R;Pansare K;McLean P;Yang J;Kolch W;Pitt AR
在定量实时聚合酶链反应（qPCR）实验中适当的标准化控制的重要性已变得更加明显，因为使用这种方法的生物学研究的数量已经增加。在开发研究瞬时转染质粒的基因表达的系统时，很明显，使用染色体编码基因的标准化是不理想的，因为它没有考虑质粒的转染效率和显著较低的表达水平。我们已经开发并验证了使用共转染质粒的qPCR标准化方法。首先确定了在本研究中使用的转录激活剂镉、地塞米松、毛喉素和佛波醇-12-肉豆蔻酸酯-13-乙酸酯存在下用于正常化的最佳染色体基因。使用geNorm、Normfinder和BestKeeper分析qPCR数据。发现每个软件应用程序对标准化对照进行不同的排名，没有明显的相关性。在该分析中包括编码海肾荧光素酶基因（Rluc）的共转染质粒，表明其计算的稳定性不如优化的染色体基因，最可能是由于较低的表达水平和转染变异性。最后，我们通过在生物条件下测试两个染色体基因（B2 M和ActB）和共转染基因（Rluc）来验证这些分析。当分析共转染的质粒时，与染色体参考基因相比，Rluc归一化给出最小的误差。我们的数据表明，转染的Rluc是用于瞬时转染qPCR分析的最合适的标准化参考基因;它显著降低了生物实验中的标准偏差，因为它考虑了转染效率并且具有容易控制的表达水平。这提高了重现性、数据有效性，最重要的是，能够准确解释qPCR数据。
The importance of appropriate normalization controls in quantitative real-time polymerase chain reaction (qPCR) experiments has become more apparent as the number of biological studies using this methodology has increased. In developing a system to study gene expression from transiently transfected plasmids, it became clear that normalization using chromosomally encoded genes is not ideal, at it does not take into account the transfection efficiency and the significantly lower expression levels of the plasmids. We have developed and validated a normalization method for qPCR using a co-transfected plasmid. The best chromosomal gene for normalization in the presence of the transcriptional activators used in this study, cadmium, dexamethasone, forskolin and phorbol-12-myristate 13-acetate was first identified. qPCR data was analyzed using geNorm, Normfinder and BestKeeper. Each software application was found to rank the normalization controls differently with no clear correlation. Including a co-transfected plasmid encoding the Renilla luciferase gene (Rluc) in this analysis showed that its calculated stability was not as good as the optimised chromosomal genes, most likely as a result of the lower expression levels and transfection variability. Finally, we validated these analyses by testing two chromosomal genes (B2M and ActB) and a co-transfected gene (Rluc) under biological conditions. When analyzing co-transfected plasmids, Rluc normalization gave the smallest errors compared to the chromosomal reference genes. Our data demonstrates that transfected Rluc is the most appropriate normalization reference gene for transient transfection qPCR analysis; it significantly reduces the standard deviation within biological experiments as it takes into account the transfection efficiencies and has easily controllable expression levels. This improves reproducibility, data validity and most importantly, enables accurate interpretation of qPCR data.
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评估合适的参考基因用于牛肌肉组织中的基因表达研究。
影响因子：
--
作者：
Pérez R;Tupac-Yupanqui I;Dunner S
通讯作者：
Dunner S
影响因子：
2.7
作者：
Dheda, K;Huggett, JF;Zumla, A
通讯作者：
Zumla, A
影响因子：
2.7
作者：
Aerts, JL;Gonzales, MI;Topalian, SL
通讯作者：
Topalian, SL
影响因子：
7
作者：
Heid, CA;Stevens, J;Williams, PM
通讯作者：
Williams, PM
DOI：
10.1016/s0006-291x(02)00601-0
发表时间：
2002-06-28
影响因子：
3.1
作者：
Matuszyk, J;Ziolo, E;Strzadala, L
通讯作者：
Strzadala, L