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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Mol. Neurosci. | doi: 10.3389/fnmol.2019.00283

Tissue specific reference genes for microRNA expression analysis in a mouse model of peripheral nerve injury

  • 1Innsbruck Medical University, Austria

MicroRNAs (miRNAs) have emerged as master switch regulators in many biological processes in health and disease, including neuropathy. miRNAs are commonly quantified by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), usually estimated as relative expression through reference genes normalization. Different non-coding RNAs (ncRNAs) are used for miRNA normalization, however, there is no study identifying the optimal reference genes in animal models for peripheral nerve injury. We evaluated the stability of eleven ncRNAs, commonly used for miRNA normalization, in dorsal root ganglia (DRG), dorsal horn of the spinal cord (dhSC) and medial prefrontal cortex (mPFC) in the mouse spared nerve injury model. After RT-qPCR, the stability of each ncRNA was determined by using four different methods: BestKeeper, the comparative delta-Cq method, geNorm and NormFinder. The candidates were rated according to their performance in each method and an overall ranking list was compiled. The most stable ncRNAs were: sno420, sno429 and sno202 in DRG; sno429, sno202 and U6 in dhSC; sno202, sno420 and sno142 in mPFC. We provide the first reference genes’ evaluation for miRNA normalization in different neuronal tissues in an animal model of peripheral nerve lesion. Our results underline the need for careful selection of reference genes for miRNA normalization in different tissues and experimental conditions. We further anticipate that our findings can be used in a broad range of nerve injury related studies, to ensure validity and promote reproducibility in miRNA quantification.

Keywords: miRNA normalization, ncRNA, spared nerve injury (SNI), Dorsal root ganglia (DRG), spinal dorsal horn (dhSC), medial prefrontal cortex (mPFC)

Received: 25 Jun 2019; Accepted: 06 Nov 2019.

Copyright: © 2019 Kalpachidou, Kummer, Mitrić and Kress. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Dr. Theodora Kalpachidou, Innsbruck Medical University, Innsbruck, Austria, tkalpachidou@gmail.com