Scripps Genomic Medicine and Scripps Translational Science Institute, The Scripps Research Institute, La Jolla, CA 92037, USA.
BMC Genomics. 2011 Aug 15;12:412. doi: 10.1186/1471-2164-12-412.
Microarray-based gene expression analysis of peripheral whole blood is a common strategy in the development of clinically relevant biomarker panels for a variety of human diseases. However, the results of such an analysis are often plagued by decreased sensitivity and reliability due to the effects of relatively high levels of globin mRNA in whole blood. Globin reduction assays have been shown to overcome such effects, but they require large amounts of total RNA and may induce distinct gene expression profiles. The Illumina whole genome DASL assay can detect gene expression levels using partially degraded RNA samples and has the potential to detect rare transcripts present in highly heterogeneous whole blood samples without the need for globin reduction. We assessed the utility of the whole genome DASL assay in an analysis of peripheral whole blood gene expression profiles.
We find that gene expression detection is significantly increased with the use of whole genome DASL compared to the standard IVT-based direct hybridization. Additionally, globin-probe negative whole genome DASL did not exhibit significant improvements over globin-probe positive whole genome DASL. Globin reduction further increases the detection sensitivity and reliability of both whole genome DASL and IVT-based direct hybridization with little effect on raw intensity correlations. Raw intensity correlations between total RNA and globin reduced RNA were 0.955 for IVT-based direct hybridization and 0.979 for whole genome DASL.
Overall, the detection sensitivity of the whole genome DASL assay is higher than the IVT-based direct hybridization assay, with or without globin reduction, and should be considered in conjunction with globin reduction methods for future blood-based gene expression studies.
基于微阵列的外周全血基因表达分析是开发各种人类疾病相关临床生物标志物组合的常用策略。然而，由于全血中球蛋白 mRNA 水平相对较高，这种分析的结果往往受到灵敏度和可靠性降低的影响。球蛋白减少测定法已被证明可以克服这些影响，但它们需要大量的总 RNA，并且可能会诱导不同的基因表达谱。Illumina 全基因组 DASL 测定法可以使用部分降解的 RNA 样本来检测基因表达水平，并且有可能在无需球蛋白减少的情况下检测到高度异质的全血样本中存在的稀有转录本。我们评估了全基因组 DASL 测定法在分析外周全血基因表达谱中的效用。
我们发现，与标准基于 IVT 的直接杂交相比，使用全基因组 DASL 可显著增加基因表达的检测。此外，球蛋白探针阴性的全基因组 DASL 与球蛋白探针阳性的全基因组 DASL 相比，并没有显著改善。球蛋白减少进一步提高了全基因组 DASL 和基于 IVT 的直接杂交的检测灵敏度和可靠性，对原始强度相关性的影响很小。基于 IVT 的直接杂交和全基因组 DASL 的总 RNA 和球蛋白减少 RNA 之间的原始强度相关性分别为 0.955 和 0.979。
总体而言，全基因组 DASL 测定法的检测灵敏度高于基于 IVT 的直接杂交测定法，无论是否减少球蛋白，并且应与未来基于血液的基因表达研究中的球蛋白减少方法结合使用。
