Functional identity of genes detectable in expression profiling assays following globin mRNA reduction of peripheral blood samples
Introduction
Gene expression profiling is an important technology for examining the molecular biology of complex human diseases. Early studies used patterns of gene expression from primary tissues to classify disease subtypes and predict clinical outcomes [1], [2]. For many human diseases, however, collection of primary tissues is not clinically warranted; therefore, peripheral blood is rapidly becoming the tissue of choice for molecular diagnostics [3], [4].
Gene expression patterns in peripheral blood are strongly dependent on methods of sample collection and processing [5]. Sporadic induction of gene expression or mRNA degradation can significantly alter gene expression profiles [6]. To avoid processing-induced changes, several blood collection systems containing RNA stabilizing reagents have been developed. Because globin mRNA accounts for ∼ 70% of all mRNA in whole blood, reduction of globin mRNA is essential for microarray and/or quantitative RT-PCR experiments. The GLOBINclear™ system removes > 95% of alpha- and beta-globin mRNA [7], which may improve expression profiling by increasing the percentage of present calls and decreasing sample-to-sample variability.
Despite the advantages of globin reduction for improving expression profiling [8], [9], little is known about the effects of globin removal on the detection of specific genes. In this study, we 1) evaluated the effects of globin reduction on gene expression profiles from peripheral blood, focusing on genes that were only detectable, as well as genes that were no longer detectable, after globin reduction across multiple samples and 2) classified affected genes into functional groups and levels of abundance. As expression profiling in whole blood is becoming an essential technique in disease research and clinical medicine, it is important to understand the impact of globin reduction on specific classes of genes in global gene expression studies.
Section snippets
Sample collection and RNA isolation
This research was conducted under protocols approved by the Windber Medical Center Institutional Review Board. Subjects voluntarily agreed to participate and gave written informed consent. Blood samples (2.5 mL) were obtained from five healthy individuals using the PAXgene™ Blood RNA System (Qiagen, Venlo, The Netherlands). PAXgene™ tubes were incubated at room temperature for 4 h following blood collection and then stored at − 80 °C. Prior to RNA isolation, tubes were removed from − 80 °C and
Results
Average recovery of total RNA following globin reduction was 76 ± 5%, comparable to the manufacturer's reported yield. A pronounced peak corresponding to globin cRNA at ∼ 700 nucleotides was present in nonglobin-depleted samples but was substantially reduced following GLOBINclear™. As a result, the number of present calls increased from an average of 9712 ± 525 genes in nonglobin-depleted samples to 12,365 ± 258 genes in globin-depleted samples, a 28 ± 5% increase in the number of detectable genes.
We
Discussion
Gene expression profiling using whole blood is an important tool in molecular diagnostics and clinical medicine, but high levels of globin mRNA may interfere with the accurate assessment of specific genes. We confirmed that globin reduction improves the detection sensitivity of Affymetrix HG U133A 2.0 arrays, permitting detection of 2652 ± 395 additional genes. The increased call rates likely reflect higher signal intensities and lower signal-to-noise ratios following globin reduction (Fig. 1).
Acknowledgments
This work was supported by the United States Department of Defense through the Henry M. Jackson Foundation for the Advancement of Military Medicine (MDA 905-00-1-0022 to CDS). Opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as representing the views of the Department of the Army or the Department of Defense.
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