title: Platelet genetic biomarker quantification: comparison of fluorescent microspheres and PCR platforms.
abstract: 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.
authors.affiliation: Department of Applied Mathematics and Statistics, State University of New York at Stony Brook, Stony Brook, New York, USA.
authors.forename: Erya
authors.initials: E
authors.lastname: Huang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhu
authors.forename: Anil
authors.initials: A
authors.lastname: Dhundale
authors.forename: Wadie F
authors.initials: WF
authors.lastname: Bahou
authors.forename: Dmitri V
authors.initials: DV
authors.lastname: Gnatenko
mesh_terms: D000328:Adult; D000368:Aged; D001792:Blood Platelets / Q000378:metabolism*; D005260:Female; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling / Q000379:methods*; D005819:Genetic Markers; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008297:Male; D008863:Microspheres; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D015345:Oligonucleotide Probes; D011237:Predictive Value of Tests; D012333:RNA, Messenger / Q000097:blood*; D015203:Reproducibility of Results; D055815:Young Adult
chemical_list: D005456:Fluorescent Dyes; D005819:Genetic Markers; D015345:Oligonucleotide Probes; D012333:RNA, Messenger