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

Abstract

Reverse transcription followed by quantitative polymerase chain reaction (qRT-PCR) is a powerful and commonly used tool for gene expression analysis. It requires the right choice of stably expressed reference genes for accurate normalization. In this work, we aimed to select the optimal reference genes for qRT-PCR normalization within different brain areas during the first week following pentylenetetrazole-induced seizures in immature (P20–22) Wistar rats. We have tested the expression stability of a panel of nine housekeeping genes: Actb, Gapdh, B2m, Rpl13a, Sdha, Ppia, Hprt1, Pgk1, and Ywhaz. Based on geometric averaging of ranks obtained by four common algorithms (geNorm, NormFinder, BestKeeper, Comparative Delta-Ct), we found that the stability of tested reference genes varied significantly between different brain regions. The expression of the tested panel of genes was very stable within the medial prefrontal and temporal cortex, and the dorsal hippocampus. However, within the ventral hippocampus, the entorhinal cortex and amygdala expression levels of most of the tested genes were not steady. The data revealed that in the pentylenetetrazole-induced seizure model in juvenile rats, Pgk1, Ppia, and B2m expression are the most stable within the medial prefrontal cortex; Ppia, Rpl13a, and Sdha within the temporal cortex; Pgk1, Ppia, and Rpl13a within the entorhinal cortex; Gapdh, Ppia, and Pgk1 within the dorsal hippocampus; Rpl13a, Sdha, and Ppia within the ventral hippocampus; and Sdha, Pgk1, and Ppia within the amygdala. Our data indicate the need for a differential selection of reference genes across brain regions, including the dorsal and ventral hippocampus.

Reference gene identification for normalisation of RT‐qPCR analysis in plasma samples of the rat middle cerebral artery occlusion model

“…The stability of reference genes could be affected by the types and regions of the samples. In the pentylenetetrazole model of acute seizures, it was found that reference gene stability rankings varied across brain regions, including different areas of the neocortex and the dorsal vs. ventral hippocampus (Schwarz et al., 2020). To date, GAPDH and β‐actin reference genes have predominantly been used as internal reference controls due to their high and constant expression levels in many different cells and tissues (Eisenberg & Levanon, 2013; Zhu et al., 2008).…”

“…In young rats with pentylenetetrazole-induced seizures, the stability of nine tested reference genes, Actb, Gapdh,B2m,Rpl13a,Sdha,Ppia,Hprt1,Pgk1 and Ywhaz,varied significantly between different brain regions (Schwarz et al, 2020). Therefore, the stability of reference genes varies with different experimental settings, tissue types and even different tissue regions (Chapman & Waldenström, 2015).…”

“…For example, let‐7a and miR‐16 are stably expressed in healthy and breast cancer tissues, but they are not suitable for the quantitative correction of miRNAs in lung cancer patients (Davoren et al., 2008; Peltier & Latham, 2008). In young rats with pentylenetetrazole‐induced seizures, the stability of nine tested reference genes, Actb, Gapdh, B2m, Rpl13a, Sdha, Ppia, Hprt1, Pgk1 and Ywhaz , varied significantly between different brain regions (Schwarz et al., 2020). Therefore, the stability of reference genes varies with different experimental settings, tissue types and even different tissue regions (Chapman & Waldenström, 2015).…”

Reference Genes across Nine Brain Areas of Wild Type and Prader-Willi Syndrome Mice: Assessing Differences in Igfbp7, Pcsk1, Nhlh2 and Nlgn3 Expression

“…Using these tools, researchers were able to select the most suitable reference genes and compile specific panels of reference genes for the normalization of expression data adapted to their respective experimental design. Resulting reference genes cover a wide spectrum of research topics, such as human brain samples from patients with neurodegenerative disease [ 41 ], mouse CNS tissue during development and injury [ 42 ], rat brain regions after induced seizures [ 43 ] and specific tissue regions, such as mouse choroid plexus [ 44 ].…”

Image_2.TIF

“…Detection of amplification products was carried out using SYBR Green 1 in the presence of ROX reference dye (Evrogen, Russia) according to the manufacturer's recommendations. Expression levels of genes of interest were normalized to the expression of the Ywhaz housekeeping gene (Schwarz et al, 2020) and calculated using the 2 −dCt equation. The sequences of the primers used are shown in Supplementary Table 1.…”