Characterization of globin RNA interference in gene expression profiling of whole-blood samples

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.

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.

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.

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.