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

Santos EA, Marques TE, Matos Hde C, Leite JP, Garcia-Cairasco N, Paçó-Larson ML, Gitaí DL - PLoS ONE (2015)

Bottom Line: However, controversial findings highlight the occurrence of unpredictable sources of variance in the experimental designs.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Biology, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceio, Alagoas, Brazil.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Temporal expression of the core clock transcripts in the hippocampus of rats.A) Relative amounts of transcripts at different ZT after normalization to Tubb2a/Rplp1. 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 Zeitgeber time (ZT).
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pone.0141121.g003: Temporal expression of the core clock transcripts in the hippocampus of rats.A) Relative amounts of transcripts at different ZT after normalization to Tubb2a/Rplp1. 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 Zeitgeber time (ZT).

Mentions: Following, we used Tubb2a/Rplp1 as normalizers to evaluate, systematically, the temporal profiling of Bmal1, Per1, Per3, Cry1, Cry2 and Clock mRNA levels in the hippocampus of rats sacrificed every 4 hours during a 24-h period. Fig 3 illustrate the temporal organization and phase relationship of the clock genes analyzed. Using both Acrophase and CircWave softwares, we observed that with exception of Clock, all genes showed a rhythmic pattern of expression, being Per1 and Cry2 those with the highest (0.469) and lowest (0.201) amplitude, respectively (Fig 3A). The comparison of the rhythms of each gene showed that Bmal1 (Φ = ZT2) is in antiphase with Per1 (Φ = ZT16), Per3 (Φ = ZT14.8) and Cry1 (Φ = ZT17.6), whereas Bmal1 peak is approximately 5 hours before acrophase of Cry2 (Φ = ZT7.6) (Fig 3B).


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

Santos EA, Marques TE, Matos Hde C, Leite JP, Garcia-Cairasco N, Paçó-Larson ML, Gitaí DL - PLoS ONE (2015)

Temporal expression of the core clock transcripts in the hippocampus of rats.A) Relative amounts of transcripts at different ZT after normalization to Tubb2a/Rplp1. 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 Zeitgeber time (ZT).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608695&req=5

pone.0141121.g003: Temporal expression of the core clock transcripts in the hippocampus of rats.A) Relative amounts of transcripts at different ZT after normalization to Tubb2a/Rplp1. 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 Zeitgeber time (ZT).
Mentions: Following, we used Tubb2a/Rplp1 as normalizers to evaluate, systematically, the temporal profiling of Bmal1, Per1, Per3, Cry1, Cry2 and Clock mRNA levels in the hippocampus of rats sacrificed every 4 hours during a 24-h period. Fig 3 illustrate the temporal organization and phase relationship of the clock genes analyzed. Using both Acrophase and CircWave softwares, we observed that with exception of Clock, all genes showed a rhythmic pattern of expression, being Per1 and Cry2 those with the highest (0.469) and lowest (0.201) amplitude, respectively (Fig 3A). The comparison of the rhythms of each gene showed that Bmal1 (Φ = ZT2) is in antiphase with Per1 (Φ = ZT16), Per3 (Φ = ZT14.8) and Cry1 (Φ = ZT17.6), whereas Bmal1 peak is approximately 5 hours before acrophase of Cry2 (Φ = ZT7.6) (Fig 3B).

Bottom Line: However, controversial findings highlight the occurrence of unpredictable sources of variance in the experimental designs.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Biology, Institute of Biological Sciences and Health, Federal University of Alagoas, Maceio, Alagoas, Brazil.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus