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.
References
[1]
Okamoto OK, Janjoppi L, Bonone FM, Pansani AP, da Silva AV, et al. (2010) Whole transcriptome analysis of the hippocampus: toward a molecular portrait of epileptogenesis. BMC Genomics 11: 230.
[2]
Pitkanen A, Lukasiuk K (2011) Mechanisms of epileptogenesis and potential treatment targets. Lancet Neurol 10: 173–186.
[3]
Wang YY, Smith P, Murphy M, Cook M (2010) Global expression profiling in epileptogenesis: does it add to the confusion? Brain Pathol 20: 1–16.
[4]
Romcy-Pereira RN, Gitai DL, Gitai LL, Leite JP, Garcia-Cairasco N, et al. (2008) [Genes and epilepsy II: differential gene expression in epilepsy]. Rev Assoc Med Bras 54: 461–466.
[5]
Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29: 23–39.
[6]
Ginzinger DG (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol 30: 503–512.
[7]
Cikos S, Koppel J (2009) Transformation of real-time PCR fluorescence data to target gene quantity. Anal Biochem 384: 1–10.
Erickson HS, Albert PS, Gillespie JW, Wallis BS, Rodriguez-Canales J, et al. (2007) Assessment of normalization strategies for quantitative RT-PCR using microdissected tissue samples. Lab Invest 87: 951–962.
[10]
Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25: 169–193.
[11]
Suzuki T, Higgins PJ, Crawford DR (2000) Control selection for RNA quantitation. Biotechniques 29: 332–337.
[12]
Cordoba EM, Die JV, Gonzalez-Verdejo CI, Nadal S, Roman B (2011) Selection of reference genes in Hedysarum coronarium under various stresses and stages of development. Anal Biochem 409: 236–243.
[13]
Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, et al. (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75: 291–295.
[14]
Schmittgen TD, Zakrajsek BA (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 46: 69–81.
[15]
Cavalheiro EA, Leite JP, Bortolotto ZA, Turski WA, Ikonomidou C, et al. (1991) Long-term effects of pilocarpine in rats: structural damage of the brain triggers kindling and spontaneous recurrent seizures. Epilepsia 32: 778–782.
[16]
Leite JP, Garcia-Cairasco N, Cavalheiro EA (2002) New insights from the use of pilocarpine and kainate models. Epilepsy Res 50: 93–103.
[17]
Li GL, Xiao B, Xie GJ (2003) [Temporal lobe epilepsy model induced by pilocarpine in rats]. Hunan Yi Ke Da Xue Xue Bao 28: 29–32.
[18]
Curia G, Longo D, Biagini G, Jones RS, Avoli M (2008) The pilocarpine model of temporal lobe epilepsy. J Neurosci Methods 172: 143–157.
[19]
Ermolinsky B, Pacheco Otalora LF, Arshadmansab MF, Zarei MM, Garrido-Sanabria ER (2008) Differential changes in mGlu2 and mGlu3 gene expression following pilocarpine-induced status epilepticus: a comparative real-time PCR analysis. Brain Res 226: 173–80.
[20]
Maurer-Morelli CV, de Vasconcellos JF, Reis-Pinto FC, Rocha Cde S, Domingues RR, et al. (2012) A comparison between different reference genes for expression studies in human hippocampal tissue. J Neurosci Methods 208: 44–47.
[21]
Pernot F, Dorandeu F, Beaup C, Peinnequin A (2010) Selection of reference genes for real-time quantitative reverse transcription-polymerase chain reaction in hippocampal structure in a murine model of temporal lobe epilepsy with focal seizures. J Neurosci Res 88: 1000–1008.
[22]
Wierschke S, Gigout S, Horn P, Lehmann TN, Dehnicke C, et al. (2010) Evaluating reference genes to normalize gene expression in human epileptogenic brain tissues. Biochem Biophys Res Commun 403: 385–390.
[23]
Chen J, Sochivko D, Beck H, Marechal D, Wiestler OD, et al. (2001) Activity-induced expression of common reference genes in individual cns neurons. Lab Invest 81: 913–916.
[24]
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: RESEARCH0034.
[25]
Andersen CL, Jensen JL, Orntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64: 5245–5250.
[26]
Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett 26: 509–515.
[27]
Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates. Florida: Academic Press.
[28]
Racine RJ (1972) Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 32: 281–294.
[29]
Pinel JP, Rovner LI (1978) Electrode placement and kindling-induced experimental epilepsy. Exp Neurol 58: 335–346.
[30]
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408.
[31]
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3: 1101–1108.
[32]
Ozbas-Gerceker F, Redeker S, Boer K, Ozguc M, Saygi S, et al. (2006) Serial analysis of gene expression in the hippocampus of patients with mesial temporal lobe epilepsy. Neuroscience 138: 457–474.
[33]
Xu Z, Xue T, Zhang Z, Wang X, Xu P, et al. (2011) Role of signal transducer and activator of transcription-3 in up-regulation of GFAP after epilepsy. Neurochem Res 36: 2208–2215.
[34]
Tang Y, Lu A, Aronow BJ, Wagner KR, Sharp FR (2002) Genomic responses of the brain to ischemic stroke, intracerebral haemorrhage, kainate seizures, hypoglycemia, and hypoxia. Eur J Neurosci 15: 1937–1952.
[35]
Lauren HB, Lopez-Picon FR, Brandt AM, Rios-Rojas CJ, Holopainen IE (2010) Transcriptome analysis of the hippocampal CA1 pyramidal cell region after kainic acid-induced status epilepticus in juvenile rats. PLoS One 5: e10733.
[36]
Steward O, Torre E, Phillips L, Trimmer P (1990) The Process of Reinnervation in the Dentate Gyrus of Adult Rats:Time Course of Increases in mRNA for Glial Fibrillary Acidic Protein J Neurosci. 10: 2373–2384.
[37]
Torres E, Lothman E, Stward O (1992) Glial response to neuronal activity: GFAP mRNA and protein levels are transiently increased in the hippocampus after seizures. Brain Res. 631: 256–264.
[38]
Holmberg K, Patterson P (2006) Leukemia inhibitory factor is a key regulator of astrocytic, microglial and neuronal responses in a low-dose pilocarpine injury model. Brain Res. 1075: 26–35.
[39]
Hammer J, Alvestad S, Osen K, Skare O, Sonnewald U, et al. (2008) Expression of Glutamine Synthetase and GlutamateDehydrogenase in the Latent Phase and Chronic Phase in the Kainate Model of Temporal Lobe Epilepsy. Glia 56: 856–868.
[40]
Turski WA, Cavalheiro EA, Bortolotto ZA, Mello LM, Schwarz M, et al. (1984) Seizures produced by pilocarpine in mice: a behavioral, electroencephalographic and morphological analysis. Brain Res. 321: 237–253.
[41]
Castro OW, Furtado MA, Tilelli CQ, Fernandes A, Pajolla GP, et al. (2012) Comparative neuroanatomical and temporal characterization of FluoroJade-positive neurodegeneration after status epilepticus induced by systemic and intrahippocampal pilocarpine in Wistar rats. Brain Res 1374: 43–55.
[42]
Fellin T, Haydon PG (2005) Do astrocytes contribute to excitation underlying seizures? Trends Mol Med 11: 530–533.
[43]
Li YQ, Xue T, Xu J, Xu ZC, Liu H, et al. (2009) ERK1/2 activation in reactive astrocytes of mice with pilocarpine-induced status epilepticus. Neurol Res 31: 1108–1114.
[44]
Garzillo C, Mello LEA (2002) Characterization of Reactive Astrocytes in the Chronic Phase of the Pilocarpine Model of Epilepsy Epilepsy. 43: 107–109.
