title: Characterization of globin RNA interference in gene expression profiling of whole-blood samples.
abstract: BACKGROUND
  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 interference of globin transcripts on profiling and the effectiveness of globin transcript mitigation by (a) defining and characterizing globin interference, (b) reproducing globin interference with synthetic transcripts, and (c) using ROC curves to measure sensitivity and specificity for a protocol for removing alpha- and beta-globin transcripts.
  
  
  METHODS
  We collected blood at 2 sites and extracted total RNA in PreAnalytiX PAXgene tubes. As a reference for characterizing interference, we supplemented aliquots of total RNA with synthesized globin transcripts and total RNA from human brain. Selected aliquots were processed with Ambion GLOBINclear to remove globin transcripts. All aliquots were labeled and hybridized to Agilent DNA microarrays by means of pooling schemes designed to quantify the mitigation of globin interference and to titrate gene expression signatures. Quantitative reverse transcription-PCR data were generated for comparison with microarray results.
  
  
  RESULTS
  Our supplementation and pooling strategy for comparing the microarray data among samples demonstrated that mitigation could reduce an interference signature of >1000 genes to approximately 200. Analysis of samples of endogenous globin transcripts supplemented with brain RNA indicated that results obtained with the GLOBINclear treatment approach those of peripheral blood mononuclear cell preparations.
  
  
  CONCLUSIONS
  We confirmed that both the absolute concentrations of globin transcripts and differences in transcript concentrations within a sample set are factors that cause globin interference (Genes Immun 2005;6:588-95). The methods and transcripts we have developed may be useful for quantitatively characterizing globin mRNA interference and its mitigation.
authors: Rosetta Inpharmatics, Merck & Co., Inc., Seattle, WA 98109, USA.
authors: Christopher
authors: C
authors: Wright
authors: Donald
authors: D
authors: Bergstrom
authors: Hongyue
authors: H
authors: Dai
authors: Matthew
authors: M
authors: Marton
authors: Mark
authors: M
authors: Morris
authors: George
authors: G
authors: Tokiwa
authors: Yanqun
authors: Y
authors: Wang
authors: Thomas
authors: T
authors: Fare
mesh_terms: D020869:Gene Expression Profiling / Q000379:methods*; D005914:Globins / Q000235:genetics*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000097:blood*; D020871:RNA Stability; D012333:RNA, Messenger / Q000097:blood; D012372:ROC Curve; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012333:RNA, Messenger; D012313:RNA; D005914:Globins