Universal Reference RNA as a standard for microarray experiments
Universal Reference RNA as a standard for microarray experiments
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DOI：
10.1186/1471-2164-5-20
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发表时间：
2004-03-09
期刊：
影响因子：
4.4
通讯作者：
Braman, J
中科院分区：
文献类型：
--
作者：
Novoradovskaya, N;Whitfield, ML;Braman, J
背景资料：获得可靠的和可重复的双色微阵列基因表达数据对于理解细胞系统上的扰动的生物学意义至关重要。微阵列设计、RNA制备和标记、杂交条件以及数据采集和分析是难以同时控制的变量。一个有用的工具，用于监测和控制内部和实验间的变化是通用参考RNA（URR），开发的目标是提供杂交信号在每个微阵列探针位置（点）。测量信号在每个点的实验RNA的比例参考RNA的目标，而不是依赖于绝对信号强度，降低变异性归一化信号输出在任何双色hybridization experiments.Results：人，小鼠和大鼠URR（UHRR，UMRR和URRR，分别）从池的RNA来自代表不同组织的个别细胞系。使用多种微阵列来确定与URR杂交并产生高于用户定义的阈值（微阵列覆盖度）的信号的点的百分比。对于所有测试的阵列，微阵列覆盖率始终大于80%。我们证实，单个细胞系贡献了自己独特的基因组URR，主张从几个细胞系的RNA池作为一个更好的配置URR，而不是一个单一的细胞系来源URR。微阵列覆盖率比较两个单独制备的批次，每个UHRR，UMRR和URRR是高度相关的（皮尔逊相关系数为0.97）。结论：本研究的结果表明，大量的合并RNA从个别细胞系可重复地制备，并具有不同的基因代表性。这种类型的参考提供了减少微阵列实验中变异的标准，并允许在实验和实验室内以及实验和实验室之间更可靠地比较基因表达数据。
Background: Obtaining reliable and reproducible two-color microarray gene expression data is critically important for understanding the biological significance of perturbations made on a cellular system. Microarray design, RNA preparation and labeling, hybridization conditions and data acquisition and analysis are variables difficult to simultaneously control. A useful tool for monitoring and controlling intra- and inter-experimental variation is Universal Reference RNA (URR), developed with the goal of providing hybridization signal at each microarray probe location ( spot). Measuring signal at each spot as the ratio of experimental RNA to reference RNA targets, rather than relying on absolute signal intensity, decreases variability by normalizing signal output in any two-color hybridization experiment.Results: Human, mouse and rat URR (UHRR, UMRR and URRR, respectively) were prepared from pools of RNA derived from individual cell lines representing different tissues. A variety of microarrays were used to determine percentage of spots hybridizing with URR and producing signal above a user defined threshold (microarray coverage). Microarray coverage was consistently greater than 80% for all arrays tested. We confirmed that individual cell lines contribute their own unique set of genes to URR, arguing for a pool of RNA from several cell lines as a better configuration for URR as opposed to a single cell line source for URR. Microarray coverage comparing two separately prepared batches each of UHRR, UMRR and URRR were highly correlated (Pearson's correlation coefficients of 0.97).Conclusion: Results of this study demonstrate that large quantities of pooled RNA from individual cell lines are reproducibly prepared and possess diverse gene representation. This type of reference provides a standard for reducing variation in microarray experiments and allows more reliable comparison of gene expression data within and between experiments and laboratories.