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>Clock</i> expression analysis in hippocampus of epileptic rats.

<p>Relative amounts of <i>Clock</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 = 4 rats in each epileptic group and 5 rats/time point in naive).</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

Relative quantities of Gfap in the hippocampus of intrahippocampal PILO injected rats using different reference genes for normalization.

<p>qRT-PCR data were normalized by eight single reference genes. The graphic shows mean levels (± SD) of Gfap transcripts in agude fase of epileptogenesis (experimental, n = 5) and controls (n = 6), * p<0.05.</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

Selection of the most suitable reference genes for circadian analysis in the hippocampus of rats.

<p>Expression stability measurements for the 8 reference genes calculated by geNorm (A) and NormFinder (B). The x-axis from left to right indicates the ranking of the genes according to their expression stability; lower values indicate higher expression stability. C) Determination of the optimal number of reference genes for normalization by geNorm. The Software calculates the normalization factor from at least two genes at which the variable V defines the pair-wise variation between two sequential normalization factors.</p

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

<p>Relative amounts of <i>Bmal</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

Primer sequences and amplification summary.

<p>*Clock, Circadian locomotor output cycle kaput; Cry1-2, Cryptochrome1-2; Per1-3, Period 1–3; Bmal1, Brain and muscle Arnt-like protein-1.</p><p>Primer sequences and amplification summary.</p