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Platelet genetic biomarker quantification: Comparison of fluorescent microspheres and PCR platforms

Journal: Thrombosis and Haemostasis
ISSN: 0340-6245
DOI: http://dx.doi.org/10.1160/TH12-04-0257
Issue: 2013: 109/2 (Feb) pp. 175-360
Pages: 337-346

Platelet genetic biomarker quantification: Comparison of fluorescent microspheres and PCR platforms

E. Huang (1), W. Zhu (1), A. Dhundale (2, 3), W. F. Bahou (4, 5, 6), D. V. Gnatenko (4, 7, 2)

(1) Department of Applied Mathematics and Statistics, State University of New York at Stony Brook, Stony Brook, New York, USA; (2) Stony Brook Biotechnology LLC, Stony Brook, New York, USA; (3) Long Island High Technology Incubator, State University of New York at Stony Brook, New York, USA; (4) Department of Medicine, State University of New York at Stony Brook, New York, USA; (5) Stony Brook Stem Cells Facility Center, Stony Brook, New York, USA; (6) Program in Genetics, State University of New York at Stony Brook, New York, USA; (7) Genomics Core Facility, State University of New York at Stony Brook, New York, USA

Keywords

mRNA, Platelets, transcript profiling, fluorescent microspheres, QuantiGene Plex 2.0 technology

Summary

The platelet transcriptome has been extensively characterised using distinct genetic profiling platforms, with evolving evidence for differential expression patterns between healthy individuals and subject cohorts with various haematologic and cardiovascular disorders. Traditional technological platforms for platelet genetic biomarker quantification have limited applicability for clinical molecular diagnostics due to inherent complexities related to RNA isolation and analysis. We have previously established the feasibility of fluorescent microspheres as a simple and reproducible strategy for simultaneous quantification of platelet mRNAs from small volume of blood using intact platelets. We now extend these observations by formally comparing in a 50-member normal cohort the cross-platform behaviour of fluorescent microspheres to the currently accepted Q-PCR standard, using a clinically relevant 15-biomarker gene subset able to discriminate among normal and thrombocytosis cohorts. When compared to Q-PCR, genetic biomarker quantification using fluorescent microspheres demonstrated lower coefficients of variation for low-abundant transcripts, better linearity in serially diluted samples, and good overall between-platform consistency via the geometric mean regression. Neither platform demonstrated age or gender effects for any of the 15 biomarkers studied. Binding site saturation for highly abundant transcripts using fluorescent microspheres can be readily eliminated using an optimal platelet number corresponding to 0.3 ml of peripheral blood, additionally applicable to thrombocytopenic cohorts. These data provide a detailed cross-platform analysis using a relevant biomarker subset, further highlighting the applicability of fluorescent microspheres as potentially superior to Q-PCR for platelet mRNA diagnostics.

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