Abstract
BackgroundMicroarray-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.ResultsWe 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.ConclusionsOverall, 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.
Highlights
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
Removing globin-specific oligos from the cDNA-mediated annealing (DASL) Assay Oligo Pool (DAP) should decrease noise associated with the high abundance of globin messenger RNA (mRNA) transcripts and potentially eliminate the necessity of globin reduction
The whole genome (WG)-DASL target preparation method significantly improved detection sensitivity compared to IVT-based direct hybridization (IVT) (p-value = 2.13 × 10-9 from an analysis of variance (ANOVA))
Summary
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. 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. The sensitivity and specificity of microarray assays using peripheral whole blood are reduced due to the relatively high proportion of globin mRNA present in total RNA, which obscures the detection of transcripts expressed at low levels in whole blood [5,6]. In order to assess the need for globin reduction with the Illumina DASL assay, we compared microarray gene expression profiles of peripheral blood total RNA and globin-reduced RNA amplified via in vitro transcription (IVT)-based direct hybridization, DASL with globin-specific oligos, and DASL without globin-specific oligos
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