Tissue Specific Reference Genes for MicroRNA Expression Analysis in a Mouse Model of Peripheral Nerve Injury

Abstract

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 (SNI) 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 injury. 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.

miRNA Expression Profiling in Subcutaneous Adipose Tissue of Monozygotic Twins Discordant for HIV Infection: Validation of Differentially Expressed miRNA and Bioinformatic Analysis

“…Some studies have indicated its unsuitability for miRNA normalization due to expression instability in various experimental conditions [ 31 , 32 , 33 ]. Conversely, in other models, U6 expression levels are very stable among the sample groups, making it a valid endogenous control [ 30 , 34 ]. Concerning the miRNAs present in the plasma, few reference data are available in support of selecting a reliable endogenous reference gene.…”

Role of Non-coding RNAs in Axon Regeneration after Peripheral Nerve Injury

“…MiRNAs are a type of endogenous regulatory ncRNAs found in eukaryotes, which are about 22 nucleotides long 26 . They are initially produced in the nucleus by RNA polymerase II as long primary transcripts (pri-miRNAs), which fold into hairpins to bind to Drosha and Dicer, the RNase III families of enzymes.…”

A novel method for producing functionalized vesicles that efficiently deliver oligonucleotides in vitro in cancer cells and in vivo in mice

“…Normalizing against U6 snRNA demonstrates the feasibility of our approach but is not ideal for the following reasons. U6 snRNA expression can vary in mouse tissues (even within the same organ), differ significantly between genders, and be affected by a mouse's age [108,[110][111][112][113]. U6 snRNA expression also likely varies greatly between mouse strains and sub-strains [114,115].…”

“…Finally, the calculation of variation between groups of candidate control genes can be accomplished with the NormFinder algorithm [171]. For the best results, these four methods can be used together to determine the optimum reference genes for miRNA quantitation, as recently shown for CNS tissues of mice [108]. Even after optimum reference ncRNA genes have been identified, control RNA gene expression should be monitored in the presence of the miRNA interest to ensure that the control genes are not significantly affected by the miRNA (i.e., ∆Cq = ~0).…”