Directly labeled mRNA produces highly precise and unbiased differential gene expression data
Directly labeled mRNA produces highly precise and unbiased differential gene expression data
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DOI：
10.1093/nar/gng013
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发表时间：
2003-02-15
影响因子：
14.9
通讯作者：
DiMeo, J
中科院分区：
文献类型：
--
作者：
Gupta, V;Cherkassky, A;DiMeo, J
基于微阵列的基因表达研究允许使用荧光标记的核酸靶标同时分析数千个基因的信使RNA（mRNA）的相对量。最常见的方法使用酶促技术，如寡聚-dT引发的逆转录以产生标记的cDNA。这些标记方法具有许多缺点，包括酶引入的标记和序列偏差、费力的方案、高实验间变异性和不能检测表达水平的小变化。在这里，我们描述了一种新的标记方法，使用铂连接的花青染料直接化学标记的mRNA从少至2杯的总RNA。我们表明，使用标记的mRNA方法产生的基因表达数据具有非常高的精度，低误差，没有标记偏差和动态范围超过几个数量级。这使得识别差异表达基因的准确性更高，并减少了运行过多重复测定的需要。现在，使用这种简单、方便和快速的方法，可以在大规模基因表达谱分析中检测基因表达的微小变化。
Microarray based gene expression studies allow simultaneous analysis of relative amounts of messenger RNA (mRNA) for thousands of genes using fluorescently labeled nucleic acid targets. Most common methods use enzymatic techniques, such as oligo-dT primed reverse transcription to produce labeled cDNA. These labeling methods have a number of shortcomings, including enzyme- introduced labeling and sequence bias, laborious protocols, high experiment-to-experiment variability and an inability to detect small changes in expression levels. Here, we describe a novel labeling methodology that uses platinum-linked cyanine dyes to directly chemically label mRNA from as little as 2 mug of total RNA. We show that the gene expression data produced using the labeled mRNA method has very high precision, low error, no labeling bias and a dynamic range over several orders of magnitude. This allows a greater accuracy in the identification of differentially expressed genes and cuts down on the need for running too many replicate assays. Small changes in gene expression can now be detected in large-scale gene expression profiling assays using this simple, easy and quick procedure.