Validation of Suitable Reference Genes for Expression Studies in Different Pilocarpine-Induced Models of Mesial Temporal Lobe Epilepsy

Abstract

It is well recognized that the reference gene in a RT-qPCR should be properly validated to ensure that gene expression is unaffected by the experimental condition. We investigated eight potential reference genes in two different pilocarpine PILO-models of mesial temporal lobe epilepsy (MTLE) performing a stability expression analysis using geNorm, NormFinder and BestKepeer softwares. Then, as a validation strategy, we conducted a relative expression analysis of the Gfap gene. Our results indicate that in the systemic PILO-model Actb, Gapdh, Rplp1, Tubb2a and Polr1a mRNAs were highly stable in hippocampus of rats from all experimental and control groups, whereas Gusb revealed to be the most variable one. In fact, we observed that using Gusb for normalization, the relative mRNA levels of the Gfap gene differed from those obtained with stable genes. On the contrary, in the intrahippocampal PILO-model, all softwares included Gusb as a stable gene, whereas B2m was indicated as the worst candidate gene. The results obtained for the other reference genes were comparable to those observed for the systemic Pilo-model. The validation of these data by the analysis of the relative expression of Gfap showed that the upregulation of the Gfap gene in the hippocampus of rats sacrificed 24 hours after status epilepticus (SE) was undetected only when B2m was used as the normalizer. These findings emphasize that a gene that is stable in one pathology model may not be stable in a different experimental condition related to the same pathology and therefore, the choice of reference genes depends on study design.

Using Postmortem hippocampi tissue can interfere with differential gene expression analysis of the epileptogenic process

“…In differential gene expression studies, two types of variation can affect the results: i) biological variation among individuals, such as age, gender, ethnicity, body mass index (BMI), lifestyle, and other individual characteristics [ 10 – 14 ]; ii) technical variation due to sample processing, such as pipetting errors, reverse transcription efficiency, RNA quality and other [ 15 , 16 ]. Several strategies can be adopted to minimize these factors, including the use of numerous biological replicates in matched-pairs design for biological variances and the use of suitable normalizers for technical variations [ 17 – 20 ]. Recognition of these confounding effects is, therefore, essential for a robust experimental design.…”

Validation of reference genes for quantitative gene expression analysis in experimental epilepsy

“…A limitation of the present study is that we did not evaluate potential reference genes in other epilepsy models (e.g., pilocarpine or intra‐amygdala kainate), in other strains, or in other model organisms. Previous studies have, however, validated some of the same reference genes both in human epilepsy samples (Wierschke et al, ) and in the pilocarpine rat model of epilepsy using male Wistar rats (Marques et al, ). Regarding the former, TBP, ACTB , and UBC were found to be suitable reference genes in human samples; we show here that they are appropriate for use in the 8‐hr and 30‐min PPS models.…”

“…The same study showed ACTB and GAPDH to be stable reference genes; these were ranked first in the KaL epileptogenesis and chronic epilepsy groups, respectively (Figure 1, Panel C and D). Marques and colleagues also found RPLP1 to be another stable reference gene in the pilocarpine model (Marques et al, 2013); it was ranked among the top four for both the 30-min PPS and chronic KaL groups (Figure 1, Panel B and D).…”

Region-specific alterations of AMPA receptor phosphorylation and signaling pathways in the pilocarpine model of epilepsy

“…3F) in chronic period may reflect the first step of a process in which seizures induce astrocytic hypertrophy and may be also related with brain damage. Changes in astrocytes can modify the homeostasis in the extracellular microenvironment contributing to the development of an epileptic state (Chen et al, 2013;Marques et al, 2013;Steward et al, 1992). The decline of EAAT2 expression (Fig.…”