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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 profile of Per1 transcripts in the hippocampus based in two different normalization strategies.RT-qPCR data were normalized by best combination of genes derived by geNorm (A,C) and NormFinder (B,D) analysis (mean, n = 5). Statistical test for circadian analysis by Acrophase (A, B) and CircWave (C, D). Data are presented as mean (n = 5 rats/time point).
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pone.0141121.g002: Temporal profile of Per1 transcripts in the hippocampus based in two different normalization strategies.RT-qPCR data were normalized by best combination of genes derived by geNorm (A,C) and NormFinder (B,D) analysis (mean, n = 5). Statistical test for circadian analysis by Acrophase (A, B) and CircWave (C, D). Data are presented as mean (n = 5 rats/time point).

Mentions: Our first aim was to identify genes that could be used as normalizers for RT-qPCR analysis in hippocampus of Wistar rats throughout a 12:12 light-dark cycle. We evaluated expression stability of the candidate genes in hippocampus samples harvest at different ZTs, using geNorm and Normfinder softwares. The average expression stability values (M values) of the reference genes in all tested samples are displayed in Fig 1A. All the genes presented high expression stability, with the M values varying between 0.21 (Ppia) and 0.46 (Gusb). The pairwise variation V2/3 was 0.069 (Fig 1C); thus, the Tubb2a/Rplp1 genes were indicated as the optimal pair to provide normalization of gene expression at the different photoperiods tested. Results of NormFinder analysis are shown in Fig 1B. Also, Ppia and Gusb appeared, as the most and the least stable genes (stability value of 0.052 and 0.46), respectively. The best combination of reference genes indicated was Tubb2a/Ppia. These data sets are comparable with those obtained using geNorm, with slight differences in the ranking order of the most stable genes and of the best pair combination. In order to validate the results obtained, we conducted a relative expression analysis of the Per1 gene. We used the recommended combination of genes from both geNorm (Tubb2a/Rplp1) and NormFinder (Tubb2a/Ppia) as internal controls. The statistical analysis of Per1 diurnal expression was performed by Acrophase and CircWave softwares. We observed that with both normalization procedures, a Per1 transcript levels oscillate in a rhythmic pattern, peaking at approximately ZT16 and with amplitude nearly of 0.47 (Fig 2).


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 profile of Per1 transcripts in the hippocampus based in two different normalization strategies.RT-qPCR data were normalized by best combination of genes derived by geNorm (A,C) and NormFinder (B,D) analysis (mean, n = 5). Statistical test for circadian analysis by Acrophase (A, B) and CircWave (C, D). Data are presented as mean (n = 5 rats/time point).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141121.g002: Temporal profile of Per1 transcripts in the hippocampus based in two different normalization strategies.RT-qPCR data were normalized by best combination of genes derived by geNorm (A,C) and NormFinder (B,D) analysis (mean, n = 5). Statistical test for circadian analysis by Acrophase (A, B) and CircWave (C, D). Data are presented as mean (n = 5 rats/time point).
Mentions: Our first aim was to identify genes that could be used as normalizers for RT-qPCR analysis in hippocampus of Wistar rats throughout a 12:12 light-dark cycle. We evaluated expression stability of the candidate genes in hippocampus samples harvest at different ZTs, using geNorm and Normfinder softwares. The average expression stability values (M values) of the reference genes in all tested samples are displayed in Fig 1A. All the genes presented high expression stability, with the M values varying between 0.21 (Ppia) and 0.46 (Gusb). The pairwise variation V2/3 was 0.069 (Fig 1C); thus, the Tubb2a/Rplp1 genes were indicated as the optimal pair to provide normalization of gene expression at the different photoperiods tested. Results of NormFinder analysis are shown in Fig 1B. Also, Ppia and Gusb appeared, as the most and the least stable genes (stability value of 0.052 and 0.46), respectively. The best combination of reference genes indicated was Tubb2a/Ppia. These data sets are comparable with those obtained using geNorm, with slight differences in the ranking order of the most stable genes and of the best pair combination. In order to validate the results obtained, we conducted a relative expression analysis of the Per1 gene. We used the recommended combination of genes from both geNorm (Tubb2a/Rplp1) and NormFinder (Tubb2a/Ppia) as internal controls. The statistical analysis of Per1 diurnal expression was performed by Acrophase and CircWave softwares. We observed that with both normalization procedures, a Per1 transcript levels oscillate in a rhythmic pattern, peaking at approximately ZT16 and with amplitude nearly of 0.47 (Fig 2).

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