A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy

Ryan J. Farr, Andrzej S. Januszewski, Mugdha V. Joglekar, Helena Liang, Annie K. McAulley, Alex W. Hewitt, Helen E. Thomas, Tom Loudovaris, Thomas W.H. Kay, Alicia Jenkins, Anandwardhan A. Hardikar

Research output: Contribution to journalArticleResearchpeer-review

40 Citations (Scopus)

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.

Original languageEnglish
Article number10375
Number of pages11
JournalScientific Reports
Volume5
DOIs
Publication statusPublished - 2 Jun 2015
Externally publishedYes

Keywords

  • Biomarkers
  • Diabetic Retinopathy/genetics*
  • Cluster Analysis
  • Diabetic Retinopathy/blood
  • Diabetic Retinopathy/pathology
  • Fluorescein Angiography
  • Gene Expression Profiling*
  • High-Throughput Nucleotide Sequencing*
  • Humans
  • MicroRNAs/blood
  • MicroRNAs/genetics*

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