The Reference Gene Validation in the Brain of Rats during Antioxidant and Anti-Inflammatory Treatment in the Lithium-Pilocarpine Model of Temporal Epilepsy

• Published in: Journal of evolutionary biochemistry and physiology Vol. 58; no. 3; pp. 930 - 940
• Main Authors: Schwarz, A. P., Dyomina, A. V., Zakharova, M. V., Kovalenko, A. A., Gryaznova, M. O., Ischenko, A. M., Zaitsev, A. V.
• Format: Journal Article
• Language: English
• Published: Moscow Pleiades Publishing 01-05-2022; Springer Nature B.V
• Subjects: Adenosylmethionine; Animal Physiology; Antioxidants; Ascorbic acid; Biochemistry; Biomedical and Life Sciences; Cortex (temporal); Cytokines; Epilepsy; Evolutionary Biology; Experimental Papers; Gene expression; Glyceraldehyde-3-phosphate dehydrogenase; Hippocampus; IL-1β; Inflammation; Interleukin 1; Interleukin 1 receptor antagonist; Interleukin 1 receptors; Life Sciences; Lithium; Pilocarpine; Reverse transcription; S-Adenosylmethionine; Seizures; Temporal lobe; Vitamin C; Vitamin E; reference genes; temporal lobe epilepsy; lithium-pilocarpine model; RT-PCR; real time PCR; mRNA; brain; gene expression
• ISSN: 0022-0930; 1608-3202
• DOI: 10.1134/S0022093022030279

Reverse transcription combined with quantitative (real-time) polymerase chain reaction (RT-qPCR) is the gold standard for assessing gene expression, but requires careful selection of valid reference genes to normalize the results. The aim of this work was to determine the optimal reference genes for RT-qPCR experiments in the temporal cortex and dorsal hippocampus of the rat brain in the latent phase (the period of epileptogenesis before the onset of spontaneous recurrent seizures) of the lithium-pilocarpine model of temporal lobe epilepsy during the separate or combined treatment with recombinant pro-inflammatory cytokine, an interleukin-1 receptor antagonist (100 mg/kg i.p. per day) and a balanced antioxidant cocktail (100 mg/kg vitamin C, 200 mg/kg vitamin E, 25 mcg/kg S-adenosylmethionine i.p. per day). The expression of 9 reference genes (
Actb
,
B2m
,
Gapdh
,
Rpl13a
,
Sdha
,
Ppia
,
Hprt1
,
Pgk1
,
Ywhaz
) was analyzed. Using the RefFinder® online tool, we determined the optimal reference genes for assessing the levels of target mRNAs in the dorsal hippocampus (
Gapdh
,
Pgk1
,
Hprt1
) and temporal cortex (
Pgk1
,
Sdha
,
Ppia
). At the same time, we revealed a significant increase in the expression of the
Actb
and
B2m
genes in the dorsal hippocampus, which was not suppressed either by the treatment with IL-1ra, or by a mixture of antioxidants (vitamin C, vitamin E, adenosylmethionine), or by their combination. We have also assessed the mRNA expression of proinflammatory cytokine interleukin-1 β (
Il1b
gene) in the dorsal hippocampus, normilising it to the three optimal (
Gapdh
,
Pgk1
,
Hprt1
) or unstable (
Actb
and
B2m
) reference genes. Using the most stable reference genes, there was a significant increase in
Il1b
gene expression levels after status epilepticus, but it was not detected when normalised to B2m or Actb gene expression. Based on this, the
Actb
and
B2m
genes may be involved in the epileptogenesis and cannot be used as reference genes in epilepsy models. Thus, the results of our work highlight the importance of careful selection of reference genes.