title: A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy.
abstract: MicroRNAs are now increasingly recognized as biomarkers of disease progression. Several quantitative real-time PCR (qPCR) platforms have been developed to determine the relative levels of microRNAs in biological fluids. We systematically compared the detection of cellular and circulating microRNA using a standard 96-well platform, a high-content microfluidics platform and two ultra-high content platforms. We used extensive analytical tools to compute inter- and intra-run variability and concordance measured using fidelity scoring, coefficient of variation and cluster analysis. We carried out unprejudiced next generation sequencing to identify a microRNA signature for Diabetic Retinopathy (DR) and systematically assessed the validation of this signature on clinical samples using each of the above four qPCR platforms. The results indicate that sensitivity to measure low copy number microRNAs is inversely related to qPCR reaction volume and that the choice of platform for microRNA biomarker validation should be made based on the abundance of miRNAs of interest.
authors.affiliation: Diabetes and Islet biology Group, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney, Level 6, Medical Foundation Building, 92-94 Parramatta Road, Camperdown, NSW 2050, Australia.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Farr
authors.affiliation: Biomarkers Laboratory, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney, Level 6, Medical Foundation Building, 92-94 Parramatta Road, Camperdown, NSW 2050, Australia.
authors.forename: Andrzej S
authors.initials: AS
authors.lastname: Januszewski
authors.affiliation: Diabetes and Islet biology Group, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney, Level 6, Medical Foundation Building, 92-94 Parramatta Road, Camperdown, NSW 2050, Australia.
authors.forename: Mugdha V
authors.initials: MV
authors.lastname: Joglekar
authors.affiliation: Clinical Genetics Unit, Center for Eye Research Australia (CERA), The University of Melbourne, Royal Victorian Eye and Ear Hospital, Peter Howson Wing, Level 1, 32 Gisborne Street, Melbourne, VIC 3002, Australia.
authors.forename: Helena
authors.initials: H
authors.lastname: Liang
authors.affiliation: Clinical Genetics Unit, Center for Eye Research Australia (CERA), The University of Melbourne, Royal Victorian Eye and Ear Hospital, Peter Howson Wing, Level 1, 32 Gisborne Street, Melbourne, VIC 3002, Australia.
authors.forename: Annie K
authors.initials: AK
authors.lastname: McAulley
authors.affiliation: Clinical Genetics Unit, Center for Eye Research Australia (CERA), The University of Melbourne, Royal Victorian Eye and Ear Hospital, Peter Howson Wing, Level 1, 32 Gisborne Street, Melbourne, VIC 3002, Australia.
authors.forename: Alex W
authors.initials: AW
authors.lastname: Hewitt
authors.affiliation: Immunology and Diabetes Unit, St. Vincent's Institute of Medical Research, 9 Princes St, Fitzroy, VIC 3065, Australia.
authors.forename: Helen E
authors.initials: HE
authors.lastname: Thomas
authors.affiliation: Immunology and Diabetes Unit, St. Vincent's Institute of Medical Research, 9 Princes St, Fitzroy, VIC 3065, Australia.
authors.forename: Tom
authors.initials: T
authors.lastname: Loudovaris
authors.affiliation: Immunology and Diabetes Unit, St. Vincent's Institute of Medical Research, 9 Princes St, Fitzroy, VIC 3065, Australia.
authors.forename: Thomas W H
authors.initials: TW
authors.lastname: Kay
authors.affiliation: Biomarkers Laboratory, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney, Level 6, Medical Foundation Building, 92-94 Parramatta Road, Camperdown, NSW 2050, Australia.
authors.forename: Alicia
authors.initials: A
authors.lastname: Jenkins
authors.affiliation: Diabetes and Islet biology Group, NHMRC Clinical Trials Centre, Faculty of Medicine, The University of Sydney, Level 6, Medical Foundation Building, 92-94 Parramatta Road, Camperdown, NSW 2050, Australia.
authors.forename: Anandwardhan A
authors.initials: AA
authors.lastname: Hardikar
mesh_terms: D015415:Biomarkers; D016000:Cluster Analysis; D003930:Diabetic Retinopathy / Q000097:blood / Q000235:genetics* / Q000473:pathology; D005451:Fluorescein Angiography; D020869:Gene Expression Profiling; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D035683:MicroRNAs / Q000097:blood / Q000235:genetics*
chemical_list: D015415:Biomarkers; D035683:MicroRNAs