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.
Conclusion: 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.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1373/clinchem.2007.093419 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
MYB represses ζ-globin expression through upregulating ETO2.
Acta Biochim Biophys Sin (Shanghai)
January 2025
Innovation Center for Diagnostics and Treatment of Thalassemia, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Reactivating the embryonic ζ-globin gene represents a potential therapeutic approach to ameliorate the severe clinical phenotype of α-thalassemia and sickle cell disease. The transcription factor MYB has been extensively proven to be a master regulator of the γ-globin gene, but its role in the regulation of ζ-globin remains incompletely understood. Here, we report a mechanistic study on the derepression of ζ-globin both and .
View Article and Find Full Text PDFOxidized phospholipid and transcriptomic signatures of THC-related vaping associated lung injury.
Sci Rep
December 2024
Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
E-cigarette/vaping-associated lung injury (EVALI) is strongly associated with vitamin E acetate and often occurs with concomitant tetrahydrocannabinol (THC) use. To uncover pathways associated with EVALI, we examined cytokines, transcriptomic signatures, and lipidomic profiles in bronchoalveolar lavage fluid (BALF) from THC-EVALI patients. At a single center, we prospectively enrolled mechanically ventilated patients with EVALI from THC-containing products (N = 4) and patients with non-vaping acute lung injury and airway controls (N = 5).
View Article and Find Full Text PDFHaplotype-Resolved Genotyping and Association Analysis of 1,020 β-Thalassemia Patients by Targeted Long-Read Sequencing.
Adv Sci (Weinh)
December 2024
Innovation Center for Diagnostics and Treatment of Thalassemia, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, 510515, China.
Despite the well-documented mutation spectra of β-thalassemia, the genetic variants and haplotypes of globin gene clusters modulating its clinical heterogeneity remain incompletely illustrated. Here, a targeted long-read sequencing (T-LRS) is demonstrated to capture 20 genes/loci in 1,020 β-thalassemia patients. This panel permits not only identification of thalassemia mutations at 100% of sensitivity and specificity, but also detection of rare structural variants (SVs) and single nucleotide variants (SNVs) in modifier genes/loci.
View Article and Find Full Text PDFEnhancing human ACE2 expression in mouse models to improve COVID-19 research.
FEBS Open Bio
December 2024
Guangzhou National Laboratory, Guangzhou, China.
Mice are one of the most common biological models for laboratory use. However, wild-type mice are not susceptible to COVID-19 infection due to the low affinity of mouse ACE2, the entry protein for SARS-CoV-2. Although mice with human ACE2 (hACE2) driven by Ace2 promoter reflect its tissue specificity, these animals exhibit low ACE2 expression, potentially limiting their fidelity in mimicking COVID-19 manifestations and their utility in viral studies.
View Article and Find Full Text PDFDomain architecture of the MabR (), a member of the PucR transcription factor family.
Heliyon
November 2024
Unit of Microbiology, Bioorganic and Macromolecular Chemistry, Department of Research in Drug Development, Faculty of Pharmacy, Université Libre de Bruxelles, Belgium.
MabR (), a PucR-type transcription factor, plays a crucial role in regulating mycolic acid biosynthesis in . To understand its regulatory mechanisms, we determined the crystal structures of its N-terminal and C-terminal domains. The N-terminal domain adopts a globin-like fold, while the C-terminal domain comprises an α/β GGDEF domain and an all-α effector domain with a helix-turn-helix DNA-binding motif.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!