A qualitative assessment of direct-labeled cDNA products prior to microarray analysis
A qualitative assessment of direct-labeled cDNA products prior to microarray analysis
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DOI：
10.1186/1471-2164-6-36
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发表时间：
2005-03-11
期刊：
影响因子：
4.4
通讯作者：
Tucker, CJ
中科院分区：
文献类型：
--
作者：
Grissom, SF;Lobenhofer, EK;Tucker, CJ
背景：微阵列过程在确定数千个基因的差异基因表达方面的成功取决于起始RNA的质量和完整性，对于通过逆转录程序进行直接标记尤其如此。此外，如果标记效率较差，质量合理的 RNA 仍可能无法产生可靠的杂交数据。 结果：在此，我们提出了一种新的测定方法，用于在使用 Agilent 2100 生物分析仪进行微阵列杂交之前评估直接标记的荧光 cDNA 的质量，该分析仪采用微流控技术来分析核酸和蛋白质。使用不同量的 RNase 来模拟 RNA 降解，我们展示了这种未公开的测定法在确定从直接标记反应中获得的 cDNA 相对量方面的优势。结论：在实验室中利用这种方法将有助于防止将劣质直接标记产品与昂贵的阵列杂交的代价高昂的错误。
Background: The success of the microarray process in determining differential gene expression of thousands of genes is dependent upon the quality and integrity of the starting RNA, this being particularly true of direct labeling via a reverse transcription procedure. Furthermore, an RNA of reasonable quality still may not yield reliable hybridization data if the labeling efficiency was poor.Results: Here we present a novel assay for assessing the quality of directly labeled fluorescent cDNA prior to microarray hybridization utilizing the Agilent 2100 Bioanalyzer, which employs microfluidic technology for the analysis of nucleic acids and proteins. Using varying amounts of RNase to simulate RNA degradation, we show the strength of this un-advertised assay in determining the relative amounts of cDNA obtained from a direct labeling reaction.Conclusion: Utilization of this method in the lab will help to prevent the costly mistake of hybridizing poor quality direct labeled products to expensive arrays.