Validation of suitable reference genes for expression studies in different pilocarpine-induced models of mesial temporal lobe epilepsy.

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

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

The molecular mechanisms underlying epileptogenesis have been widely investigated by differential gene expression approach, especially RT-qPCR methodology. However, controversial findings highlight the occurrence of unpredictable sources of variance in the experimental designs. Here, we investigated if diurnal rhythms of transcript's levels may impact on differential gene expression analysis in hippocampus of rats with experimental epilepsy. For this, we have selected six core clock genes (Per1, Per3, Bmal1, Clock, Cry1 and Cry2), whose rhythmic expression pattern in hippocampus had been previously reported. Initially, we identified Tubb2a/Rplp1 and Tubb2a/Ppia as suitable normalizers for circadian studies in hippocampus of rats maintained to 12:12 hour light:dark (LD) cycle. Next, we confirmed the temporal profiling of Per1, Per3, Bmal1, Cry1 and Cry2 mRNA levels in the hippocampus of naive rats by both Acrophase and CircWave statistical tests for circadian analysis. Finally, we showed that temporal differences of sampling can change experimental results for Per1, Per3, Bmal1, Cry1 and Cry2, but not for Clock, which was consistently decreased in rats with epilepsy in all comparison to the naive group. In conclusion, our study demonstrates it is mandatory to consider diurnal oscillations, in order to avoid erroneous conclusions in gene expression analysis in hippocampus of rats with epilepsy. Investigators, therefore, should be aware that genes with circadian expression could be out of phase in different animals of experimental and control groups. Moreover, our results indicate that a sub-expression of Clock may be involved in epileptogenicity, although the functional significance of this remains to be investigated

Relative quantities of Gfap in the hippocampus of systemic PILO injected rats upon different normalization approaches.

<p>qRT-PCR data were normalized by eight single reference genes and best combination derived by geNorm or NormFinder (mean ± SD), n = 6. The diagram shows mean levels of Gfap transcripts in epileptogenesis (0 h and 24 h), chronic period and animal naives. *24 h compared with 0h or naive group, p<0.05; ***24 h compared with naive group, p<0.05.</p

Primer sequences and amplification summary.

*<p><i>Tubb2a</i>, tubulin beta 2A class IIa; <i>B2m</i>, β-2-Microglobulin; <i>Actb</i>, β-Actin; <i>Gapdh</i>, Glyceraldehyde-3-phospate dehydrogenase; <i>Gusb</i>, β-Glucuronidase; <i>Ppia</i>, peptidylprolyl isomerase A; <i>Porla1a</i>, polymerase (RNA) I polypeptide A; <i>Rplp1</i>, ribosomal protein, large, P1; <i>Gfap</i>, glial fibrillary acidic protein.</p

Expression levels of the candidate reference genes across experimental samples.

<p>Values are given in the form of RT-qPCR threshold cycle numbers (Ct values), mean ± SD (n = 6), * 0 h compared with 24 h, p<0.05.</p

Impact of diurnal variation on <i>Per3</i> expression analysis in hippocampus of epileptic rats.

<p>Relative amounts of <i>Per3</i>, transcripts in epileptic rats ZT08 (A) and ZT12 (B) after normalization to <i>Tubb2a</i>/<i>Rplp1</i>. Significant differences were evaluated using Unpaired Student’s t-test comparing results between epileptic and each ZT of naive group. *p<0.05, **p<0.01 and ***p<0.001. Data are presented as mean+SEM (n = 5 (ZT8) and 4 (ZT12) rats in epileptic group and n = 5 rats/time point in naive).</p

Temporal expression of the core clock transcripts in the hippocampus of rats.

<p>A) Relative amounts of transcripts at different ZT after normalization to <i>Tubb2a</i>/<i>Rplp1</i>. Data are presented as mean (n = 5 rats/ZT). Statistical test for circandian analysis by Acrophase (left) and CirWawe (right). B) Overlap of cosine fitting curves illustrating the phase relation of clock transcripts. For clarity reasons, data are doubleblotted against <i>Zeitgeber time</i> (ZT).</p

NormFinder and BestKeeper analysis of expression stability.

<p>Ranking of candidate reference genes based on stability values calculated by NormFinder (a) and BestKeeper (b) softwares for systemic PILO-model samples.</p