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

doi:10.1038/srep10375

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

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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 language	English (US)
Article number	10375
Journal	Scientific reports
Volume	5
DOIs	https://doi.org/10.1038/srep10375
State	Published - Jun 2 2015
Externally published	Yes

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Farr, R. J., Januszewski, A. S., Joglekar, M. V., Liang, H., McAulley, A. K., Hewitt, A. W., Thomas, H. E., Loudovaris, T., Kay, T. W. H., Jenkins, A., & Hardikar, A. A. (2015). A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy. Scientific reports, 5, Article 10375. https://doi.org/10.1038/srep10375

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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.",

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

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AU - Januszewski, Andrzej S.

AU - Joglekar, Mugdha V.

AU - Liang, Helena

AU - McAulley, Annie K.

AU - Hewitt, Alex W.

AU - Thomas, Helen E.

AU - Loudovaris, Tom

AU - Kay, Thomas W.H.

AU - Jenkins, Alicia

AU - Hardikar, Anandwardhan A.

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AB - 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.

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

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