Limits...
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

Impact of diurnal variation on Cry1 expression analysis in hippocampus of epileptic rats.Relative amounts of Cry1, transcripts in epileptic rats ZT08 (A) and ZT12 (B) after normalization to Tubb2a/Rplp1. 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).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608695&req=5

pone.0141121.g007: Impact of diurnal variation on Cry1 expression analysis in hippocampus of epileptic rats.Relative amounts of Cry1, transcripts in epileptic rats ZT08 (A) and ZT12 (B) after normalization to Tubb2a/Rplp1. 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).

Mentions: To investigate if the diurnal expression could be a source of variability on differential gene expression analysis in hippocampus of epileptic rats, we compared expression levels of the clock genes between epileptic rats sacrificed at ZT8 or ZT12 with naive rats sacrificed at different Zeitgeber times. In relation to ZT8 epileptic rats, clock transcripts were significantly decreased in the hippocampus of epileptic rats in all comparisons with the naive group (Fig 4A). However, the Per3 was decreased only when compared with naive rats correspondent to ZT points of the dark phase (Fig 5A). Bmal1 transcripts were significantly increased in epileptic rats only when compared with naive rats relative to the combination of dark phase ZTs (Fig 6A). Significant differences in Cry1 (increase) and Cry2 (decrease) transcripts were observed only in comparisons using specifics ZTs of light phases (Figs 7A and 8A). Intriguingly, Per1 transcripts levels were increased or decreased in hippocampus of epileptic rats depending on whether they were compared with specific ZT points of light and dark phases, respectively (Fig 9A).


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)

Impact of diurnal variation on Cry1 expression analysis in hippocampus of epileptic rats.Relative amounts of Cry1, transcripts in epileptic rats ZT08 (A) and ZT12 (B) after normalization to Tubb2a/Rplp1. 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).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141121.g007: Impact of diurnal variation on Cry1 expression analysis in hippocampus of epileptic rats.Relative amounts of Cry1, transcripts in epileptic rats ZT08 (A) and ZT12 (B) after normalization to Tubb2a/Rplp1. 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).
Mentions: To investigate if the diurnal expression could be a source of variability on differential gene expression analysis in hippocampus of epileptic rats, we compared expression levels of the clock genes between epileptic rats sacrificed at ZT8 or ZT12 with naive rats sacrificed at different Zeitgeber times. In relation to ZT8 epileptic rats, clock transcripts were significantly decreased in the hippocampus of epileptic rats in all comparisons with the naive group (Fig 4A). However, the Per3 was decreased only when compared with naive rats correspondent to ZT points of the dark phase (Fig 5A). Bmal1 transcripts were significantly increased in epileptic rats only when compared with naive rats relative to the combination of dark phase ZTs (Fig 6A). Significant differences in Cry1 (increase) and Cry2 (decrease) transcripts were observed only in comparisons using specifics ZTs of light phases (Figs 7A and 8A). Intriguingly, Per1 transcripts levels were increased or decreased in hippocampus of epileptic rats depending on whether they were compared with specific ZT points of light and dark phases, respectively (Fig 9A).

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