Jinny Liu, Elizabeth Walter, David Stenger, Dzung Thach

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Presentation transcript:

Effects of Globin mRNA Reduction Methods on Gene Expression Profiles from Whole Blood Jinny Liu, Elizabeth Walter, David Stenger, Dzung Thach The Journal of Molecular Diagnostics Volume 8, Issue 5, Pages 551-558 (November 2006) DOI: 10.2353/jmoldx.2006.060021 Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Diagram summarizing the experimental design, sample treatments, and important modifications. See the Materials and Methods section for corresponding details on sample collection and processing; total RNA isolation, cleanup, and concentration; and globin reduction and target preparation. The Journal of Molecular Diagnostics 2006 8, 551-558DOI: (10.2353/jmoldx.2006.060021) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 The globin reduction techniques efficiently reduce globin overabundance from cRNA derived from cell line or PAXgene RNA. Electropherograms of cRNA derived from Jurkat, Jurkat-globin, or PAXgene blood RNA that underwent various technical treatments. A: Traces of cRNA derived from Jurkat-globin RNA treated with DNA capture oligo (JGD), with PNA inhibitory oligo (JGP), or no treatment control (JGC), and from Jurkat RNA with no treatment control (JC). B: Traces of cRNA derived from PAXgene RNA treated with DNA capture oligo (BD), PNA inhibitory oligo (BP), or no treatment control (BC). The Journal of Molecular Diagnostics 2006 8, 551-558DOI: (10.2353/jmoldx.2006.060021) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 The globin reduction techniques affect on transcript signal variation is dependent on RNA type. The overabundance of globin mRNA mitigates the increase in transcript signal variation because of the additional steps required for globin reduction. Coefficient of variation versus signals from all probe sets for various RNA types with different technical treatments. Curves shown are from Loess 2-degree freedom fitting of the signal data points. A: Cell line RNA. Jurkat (J) or Jurkat-globin (JG) RNA treated with capture DNA oligo (JD, JGD), inhibitory PNA oligo (JP, JGP), or no globin reduction control (JC, JGC). B: PAXgene blood RNA treated with capture DNA oligo (BD), inhibitory PNA oligo (BP), or no treatment control (BC). The Journal of Molecular Diagnostics 2006 8, 551-558DOI: (10.2353/jmoldx.2006.060021) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Different treatments induced distinct gene expression profiles, but globin reduction brought the profiles closer to the original profile than without treatment, particularly with the capture oligo method. Unsupervised cluster analysis of gene expression profiles from various RNA types with different technical treatments. Logged signals from all probe sets were used. A and B: Multidimensional scaling of unscaled signals. C and D: Hierarchical clustering using center correlation and average linkage. In A and C cell line RNA, Jurkat (J) or Jurkat-globin (JG) RNA were treated with capture DNA oligo (JD, JGD), inhibitory PNA oligo (JP, JGP), or no globin reduction control (JC, JGC). In B and D, PAXgene blood RNA was treated with capture DNA oligo (BD), inhibitory PNA oligo (BP), or no treatment control (BC). The Journal of Molecular Diagnostics 2006 8, 551-558DOI: (10.2353/jmoldx.2006.060021) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 5 Patterns of changes in differential transcript signals induced by RNA types and treatment methods. Heat-maps of probe sets with differing signals among groups. Probe sets and samples were clustered using center correlation and average linkage. Only dendrograms from clustering of samples are shown on top of each panel. To select from 54,120 probe sets for those with differing signals, analysis of variance with random variance model was performed using P values ≤0.001 as significant. A: Heat map and sample clustering dendrogram of 8614 probe sets with significant differences in signals among the cell line RNA groups. Jurkat (J) or Jurkat-globin (JG) RNA treated with capture DNA oligo (JD, JGD), inhibitory PNA oligo (JP, JGP), or no globin reduction control (JC, JGC). I to V based on JGD expression pattern. B: Heat map and sample clustering dendrogram of 1988 probe sets with differing in signals among the PAXgene blood RNA groups treated with capture DNA oligo (BD), inhibitory PNA oligo (BP), or no treatment control (BC). The Journal of Molecular Diagnostics 2006 8, 551-558DOI: (10.2353/jmoldx.2006.060021) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions