Identification of Endogenous Reference Genes for the Analysis of microRNA Expression in the Hippocampus of the Pilocarpine-Induced Model of Mesial Temporal Lobe Epilepsy

Abstract

Real-time quantitative RT-PCR (qPCR) is one of the most powerful techniques for analyzing miRNA expression because of its sensitivity and specificity. However, in this type of analysis, a suitable normalizer is required to ensure that gene expression is unaffected by the experimental condition. To the best of our knowledge, there are no reported studies that performed a detailed identification and validation of suitable reference genes for miRNA qPCR during the epileptogenic process. Here, using a pilocarpine (PILO) model of mesial temporal lobe epilepsy (MTLE), we investigated five potential reference genes, performing a stability expression analysis using geNorm and NormFinder softwares. As a validation strategy, we used each one of the candidate reference genes to measure PILO-induced changes in microRNA-146a levels, a gene whose expression pattern variation in the PILO injected model is known. Our results indicated U6SnRNA and SnoRNA as the most stable candidate reference genes. By geNorm analysis, the normalization factor should preferably contain at least two of the best candidate reference genes (snoRNA and U6SnRNA). In fact, when normalized using the best combination of reference genes, microRNA-146a transcripts were found to be significantly increased in chronic stage, which is consistent with the pattern reported in different models. Conversely, when reference genes were individually employed for normalization, we failed to detect up-regulation of the microRNA-146a gene in the hippocampus of epileptic rats. The data presented here support that the combination of snoRNA and U6SnRNA was the minimum necessary for an accurate normalization of gene expression at the different stages of epileptogenesis that we tested.

Diurnal Variation Has Effect on Differential Gene Expression Analysis in the Hippocampus of the Pilocarpine-Induced Model of Mesial Temporal Lobe Epilepsy

“…Studies that aim to measure transcript levels, particularly using RT-qPCR, must use proper reference genes to avoid erroneous conclusions [ 5 , 38 , 40 ]. To the best of our knowledge, this is the first study that investigated, systematically, suitable reference genes for circadian analysis in hippocampus of Wistar rats.…”

MicroRNA profiles in hippocampal granule cells and plasma of rats with pilocarpine-induced epilepsy – comparison with human epileptic samples

“…Analysis was performed by the comparative delta threshold cycle (ΔCT) method 58 . For brain samples, U6 and snoRNA were used as reference genes 59 . For plasma, data normalization was obtained using the synthetic spiked-in c. elegans miR-39.…”

MiR‐193b regulates early chondrogenesis by inhibiting the TGF‐beta2 signaling pathway

“…RNA extraction, reverse transcription and qPCR were performed as described in Supplementary materials. The fold differences in gene expression were normalized to RNU6, HPRT and GAPDH for miRNA and mRNA [31–33], and then were calculated using the ΔΔCt method.…”