Characterization of Globin RNA Interference in Gene Expression Profiling of Whole-Blood Samples

• DOI: 10.1373/clinchem.2007.093419
• Publication Date: 2007-12-19T04:08:24Z
• Authors: Christopher Wright; Donald Bergstrom; Hongyue Dai; Matthew Marton; Mark Morris; George Tokiwa; Yanqun Wang; Thomas Fare
• Keywords: 0303 health sciences; 03 medical and health sciences; ROC Curve; Reverse Transcriptase Polymerase Chain Reaction; Gene Expression Profiling; RNA Stability; Humans; RNA; Reproducibility of Results; RNA, Messenger; Globins; Oligonucleotide Array Sequence Analysis

Blood-based biomarker discovery with gene expression profiling has been hampered by interference from endogenous, highly abundant alpha- and beta-globin transcripts. We describe a means to quantify the of globin transcripts on effectiveness transcript mitigation (a) defining characterizing interference, (b) reproducing synthetic transcripts, (c) using ROC curves measure sensitivity specificity for protocol removing transcripts.We collected blood at 2 sites extracted total RNA in PreAnalytiX PAXgene tubes. As reference we supplemented aliquots synthesized human brain. Selected were processed Ambion GLOBINclear remove All labeled hybridized Agilent DNA microarrays pooling schemes designed titrate signatures. Quantitative reverse transcription-PCR data generated comparison microarray results.Our supplementation strategy comparing among samples demonstrated that could reduce an signature >1000 genes approximately 200. Analysis endogenous brain indicated results obtained treatment approach those peripheral mononuclear cell preparations.We confirmed both absolute concentrations differences within sample set are factors cause (Genes Immun 2005;6:588-95). The methods have developed may be useful quantitatively mRNA its mitigation.