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Analytics of Cerebrospinal Fluid MicroRNA Quantitative PCR Studies

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Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that regulate post-transcriptional gene expression. Recent studies have shown that human disease states correlate with measurable differences in the level of circulating miRNAs relative to healthy controls. Thus, there is great interest in developing clinical miRNA assays as diagnostic or prognostic biomarkers for diseases, and as surrogate measures for therapeutic outcomes. Our studies have focused on miRNAs in human cerebral spinal fluid (CSF) as biomarkers for central nervous system (CNS) diseases. Our objective here was to examine factors that may affect the outcome of quantitative PCR (qPCR) studies on CSF miRNAs, in order to guide planning and interpretation of future CSF miRNA TaqMan® low-density array (TLDA) studies. We obtained CSF from neurologically normal (control) donors and used TLDAs to measure miRNA expression. We examined sources of error in the TLDA outcomes due to (1) nonspecific amplification of products in total RNA, (2) variations in RNA isolations performed on different days, (3) miRNA primer probe efficiency, and (4) variations in individual TLDA cards. We also examined the utility of card-to-card TLDA corrections and use of an unchanged “reference standard” to remove batch processing effects in large-scale studies.

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Acknowledgements

We thank the OHSU Gene Profiling Shared Resource for professional advice and access to core instrumentation. The research reported in this publication was supported by funds awarded to JAS by the Oregon Clinical & Translational Research Institute, which is supported by the NIH National Center for Advancing Translational Sciences grant UL1TR000128, by NIH NCATS UH2/UH3TR000903 (JAS, JFQ), and by the NIH National Institutes of Aging P30AG008017.

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Authors and Affiliations

  1. Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, Portland, OR, USA

    Theresa A. Lusardi

  2. Biostatistics & Design Program, Oregon Health & Science University, Portland, OR, USA

    Jack T. Wiedrick & Jodi A. Lapidus

  3. Department of Anesthesiology & Perioperative Medicine, Oregon Health & Science University, Portland, OR, USA

    Molly Malone, Jay I. Phillips, Ursula S. Sandau & Julie A. Saugstad

  4. Department of Neurology, Layton Aging and Alzheimer’s Center, Oregon Health & Science University, Portland, OR, USA

    Babett Lind & Joseph F. Quinn

  5. Portland VA Medical Center, Portland, OR, USA

    Joseph F. Quinn

Authors
  1. Theresa A. Lusardi
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  2. Jack T. Wiedrick
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  4. Jay I. Phillips
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  7. Joseph F. Quinn
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  9. Julie A. Saugstad
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Correspondence to Julie A. Saugstad.

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Lusardi, T.A., Wiedrick, J.T., Malone, M. et al. Analytics of Cerebrospinal Fluid MicroRNA Quantitative PCR Studies. Mol Neurobiol 56, 4988–4999 (2019). https://doi.org/10.1007/s12035-018-1422-0

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  • DOI: https://doi.org/10.1007/s12035-018-1422-0

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