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首页> 外文期刊>BMC Genomics >Gene expression profiling of whole blood: Comparison of target preparation methods for accurate and reproducible microarray analysis
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Gene expression profiling of whole blood: Comparison of target preparation methods for accurate and reproducible microarray analysis

机译：全血的基因表达谱：用于精确和可再现的微阵列分析的靶标制备方法的比较

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Background Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system. Results We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples. Conclusion RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the sensitivity of the DNA microarray expression profiling assay for whole blood samples. While blockage of globin transcripts during first strand cDNA synthesis with globin PNAs resulted in the best overall performance in this study, we conclude that selection of a protocol for expression profiling studies in blood should depend on several factors, including implementation requirements of the method and study design. RNA isolated from either freshly collected or frozen blood samples stored in PAXGene tubes can be used without altering gene expression profiles.
机译：背景技术外周血是许多人类疾病和药物基因组学研究的转录信息的一种可获取的信息来源。尽管分析从全血中分离的RNA可能具有显着的优势，尤其是在临床研究中，但由于需要最大限度地减少与高度丰富的球蛋白RNA转录物相关的伪影，因此用于微阵列分析的样品制备非常复杂。球蛋白RNA转录本对表达谱数据的影响可以通过使用在微阵列分析过程中去除或阻断球蛋白RNA的RNA制备和标记方法来减少。我们比较了从PAXGene管中收集的人全血中制备微阵列杂交靶标的四种不同方法。其中三种方法利用了Affymetrix单周期cDNA合成/体外转录方案，但对输入RNA的处理如下：没有治疗; ii。用GLOBINclear治疗;或iii。球蛋白PNA寡核苷酸治疗。在第四种方法中，使用Ovation扩增和标记系统制备cDNA靶标。结果我们发现，与标准Affymetrix方法相比，通过标记方法产生的微阵列靶标可降低球蛋白mRNA水平或使杂交期间球蛋白转录物的影响最小化，从而在微阵列测定中检测到更多的转录物。微阵列结果与NF-κB通路中一组基因的定量PCR分析的比较显示，尽管观察到方法相关的总体相关差异，但使用所有四种靶标制备方法产生的转录物测量值均具有良好的相关性。还检查了PAXGene管中收集的冷冻血液对数据重现性的影响。从新鲜或冷冻血液样本中提取RNA时，表达谱显示几乎没有差异。结论旨在减少球蛋白mRNA转录物影响的RNA制备和标记方法可以显着提高全血样品DNA芯片表达谱分析的灵敏度。虽然在这项研究中，在用珠蛋白PNA合成第一链cDNA时，珠蛋白转录物的阻断导致了最佳的整体性能，但我们得出的结论是，在血液中进行表达谱研究的方案的选择应取决于几个因素，包括方法和研究的实施要求设计。从PAXGene管中新鲜采集或冷冻的血液样品中分离的RNA可以使用，而无需改变基因表达谱。

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