Abstract

Non-infectious inflammatory (NII) central nervous system (CNS) conditions are primarily diagnosed by the demonstration of inflammatory changes in the cerebrospinal fluid (CSF). However, less-invasive methods and peripheral biomarkers are desired. Changes in circulating microRNA (miRNA), which are short non-coding regulatory RNAs, may serve as biomarkers of disease. The aim of this pilot study was to investigate selected miRNAs in serum and CSF, hypothesizing that the levels of specific miRNAs in serum correlate with their presence in CSF, and that changes in serum miRNAs levels may reflect CNS disease. We profiled serum and CSF samples using quantitative real-time PCR (qPCR) searching for selected and previously profiled miRNAs in serum (let-7a, let-7c, miR-15b, miR-16, miR-21, miR-23a, miR-24, miR-26a, miR-146a, miR-155, miR-181c and miR-221-3p) and in CSF (let-7c, miR-16, miR-21, miR-24, miR-146a, miR-155, miR-181c and miR-221-3p) from 13 dogs with NII CNS disease and six control dogs. We demonstrated the presence of several miRNAs in CSF (let-7c and miR-21 dominating) and serum (miR-23a and miR-21 dominating). However, we generally failed to reproduce consistent results in CSF samples due to several reasons: unacceptable PCR efficiency, a wide variation between cDNA replicates and/or no-amplification in qPCR suggesting very low levels of the investigated miRNAs in canine CSF. Serum samples performed better, and 10 miRNAs qPCR assays were qualified for analysis. We were nevertheless unable to detect a difference in the expression of miRNA levels between cases and controls. Moreover, we could not confirm the results of recent miRNA investigations of canine CNS diseases. We believe that these disagreements highlight the significant effect of methodological/analytical variation, rather than the incapacity of circulating miRNAs as biomarkers of CNS disease. A secondary aim was therefore to communicate methodological challenges in our study and to suggest recommendations for circulating miRNA profiling, including pre-, post- and analytical methods based on our experience, in order to reach reproducible and comparable results in veterinary miRNA research.

Highlights

  • Non-infectious inflammatory (NII) central nervous system (CNS) conditions are primarily diagnosed by the demon‐ stration of inflammatory changes in the cerebrospinal fluid (CSF)

  • While miRNA profiling in CSF has been successfully conducted in humans [2–5], to our knowledge only three studies in dogs are available [6–8]

  • Our hypothesis was that the levels of specific miRNAs in serum correlate with their levels in CSF and can reflect CNS inflammation

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Summary

Introduction

Non-infectious inflammatory (NII) central nervous system (CNS) conditions are primarily diagnosed by the demon‐ stration of inflammatory changes in the cerebrospinal fluid (CSF). While miRNA profiling in CSF has been successfully conducted in humans [2–5], to our knowledge only three studies in dogs are available [6–8]. A secondary aim was based on our experience, to suggest best-practice methods for future studies of miRNA profiling in dogs.

Results
Conclusion
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