The validation of Short Interspersed Nuclear Elements (SINEs) as a RT-qPCR normalization strategy in a rodent model for temporal lobe epilepsy

Abstract

BackgroundIn gene expression studies via RT-qPCR many conclusions are inferred by using reference genes. However, it is generally known that also reference genes could be differentially expressed between various tissue types, experimental conditions and animal models. An increasing amount of studies have been performed to validate the stability of reference genes. In this study, two rodent-specific Short Interspersed Nuclear Elements (SINEs), which are located throughout the transcriptome, were validated and assessed against nine reference genes in a model of Temporal Lobe Epilepsy (TLE). Two different brain regions (i.e. hippocampus and cortex) and two different disease stages (i.e. acute phase and chronic phase) of the systemic kainic acid rat model for TLE were analyzed by performing expression analyses with the geNorm and NormFinder algorithms. Finally, we performed a rank aggregation analysis and validated the reference genes and the rodent-specific SINEs (i.e. B elements) individually via Gfap gene expression.ResultsGeNorm ranked Hprt1, Pgk1 and Ywhaz as the most stable genes in the acute phase, while Gusb and B2m were ranked as the most unstable, being significantly upregulated. The two B elements were ranked as most stable for both brain regions in the chronic phase by geNorm. In contrast, NormFinder ranked the B1 element only once as second best in cortical tissue for the chronic phase. Interestingly, using only one of the two algorithms would have led to skewed conclusions. Finally, the rank aggregation method indicated the use of the B1 element as the best option to normalize target genes, independent of the disease progression and brain region. This result was supported by the expression profile of Gfap.ConclusionIn this study, we demonstrate the potential of implementing SINEs -notably the B1 element- as a stable normalization factor in a rodent model of TLE, independent of brain region or disease progression.

Peer Review #1 of "Validation of endogenous reference genes in rat cerebral cortex for RT-qPCR analyses in developmental toxicity studies (v0.1)"

“…According to three different algorithms designed to calculate relative expression stability -geNorm, NormFinder and BestKeeper -all of the 10 RGs obtained stability scores well inside recommended cut-off values, in essence predicting all of the RGs to be suitable for RT-qPCR normalization. This is in contrast to previous studies testing across different rat tissues (Svingen et al, 2015) or brain tissue from rats (Cook et al, 2009;Yang et al, 2008;Zhou et al, 2016), mice (Crans et al, 2019) or humans (Rydbirk et al, 2016). Notably, brain tissue from a rat sleep apnea model displayed high stability of all tested RGs except 18S rRNA (Julian et al, 2014), a stability also seen in rat brains across fetal developmental stages (Al-Bader & Al-Sarraf, 2005), which agrees with our findings of relative high stability among RGs in this tissue (Julian et al, 2014).…”

Reference Gene Validation in the Brain Regions of Young Rats after Pentylenetetrazole-Induced Seizures

“…The most stably expressed genes across the brain regions tested in our study were Ppia and Pgk1. The hippocampal mRNA expressions of Ppia and Pgk1 were also reported to be stable in the asphyxial cardiac arrest model and the latent phase of the kainate epilepsy model [7,15]. Ppia expression was stable in the dentate gyrus in the febrile seizure model [2].…”

“…We could not find in PubMed any previous study analyzing gene stability in the brain using the PTZ model. Only a few works investigated the stability of housekeeping gene expression using other animal models of seizure or epilepsy: after neonatal (P10) febrile seizures [2]; after short (30 min) and long (8 h) perforant pathway stimulation [15]; after systemic/intrahippocampal pilocarpine injection [32]; and in the kainic acid model of temporal lobe epilepsy [7,15]. These works analyzed the entire hippocampus [15,32] or dental gyrus of the hippocampus [2]; an exception was the study by Crans et al (2019) [7], where expression stability was analyzed in the hippocampal tissue and in the neocortex.…”

“…Different algorithms provide slightly different results [2,14,15]. To escape that bias, this paper uses a novel approach by calculating a comprehensive reference gene stability ranking based on four algorithms [3,7,[15][16][17][18][19]. Among different reference genes, Gapdh, Actb, Ppia, B2m, Hprt1, Ywhaz, Rpl13a, Pgk1, and Sdha are widely used in RT-qPCR experiments on brain tissue or brain-derived cell lines in laboratory rats.…”

“…The selection of unstable housekeeping genes as references could affect accurate quantification, leading to inconsistent results. Normalization using unstable reference genes could hide changes in target mRNA expression or find irrelevant effects of experimental treatments [ 2 , 3 , 4 , 5 , 6 , 7 , 8 ]. In the rat valproic acid model of autism spectrum disorders, changes of Pgrn1 mRNA expression in the hippocampus and cortex can have opposite directions depending on reference gene choice [ 3 ].…”