Open AccessIdentification of optimal reference genes for gene expression studies in a focal cerebral ischaemia model—Spatiotemporal effects
Author(s) -
Pomierny Bartosz,
Krzyzanowska Weronika,
Jurczyk Jakub,
Strach Beata,
Skorkowska Alicja,
Leonovich Innesa,
Budziszewska Bogusława,
Pera Joanna
Publication year - 2022
Publication title -
journal of cellular and molecular medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.44
H-Index - 130
eISSN - 1582-4934
pISSN - 1582-1838
DOI - 10.1111/jcmm.17284
Subject(s) - biology , glyceraldehyde 3 phosphate dehydrogenase , reference genes , gene expression , microbiology and biotechnology , gene , hypoxanthine guanine phosphoribosyltransferase , biochemistry , mutant
A proper reference gene (RG) is required to reliably measure mRNA levels in biological samples via quantitative reverse transcription PCR (RT‐qPCR). Various experimental paradigms require specific and stable RGs. In studies using rodent models of brain ischaemia, a variety of genes, such as β‐actin ( Actb ), hypoxanthine phosphoribosyltransferase 1 ( Hprt1 ), peptidyl‐propyl isomerase A ( Ppia ) and glyceraldehyde‐3‐phosphate dehydrogenase ( Gapdh ), are used as RGs. However, most of these genes have not been validated in specific experimental settings. The aim of this study was to evaluate the time‐ and brain region‐dependent expression of RG candidates in a rat model of transient middle cerebral artery occlusion (tMCAO). The following genes were selected: Actb , Hprt1 , Ppia , Gapdh , tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein, zeta ( Ywhaz ) and beta‐2 microglobulin ( B2m ). Focal cerebral ischaemia was induced by 90 min of tMCAO in male Sprague‐Dawley rats. Expression was investigated at four time points (12 and 24 h; 3 and 7 days) and in three brain areas (the frontal cortex, hippocampus and dorsal striatum) within the ischaemic brain hemisphere. The RT‐qPCR results were analysed using variance analysis and the ΔCt, GeNorm, NormFinder and BestKeeper methods. Data from these algorithms were ranked using the geometric mean of ranks of each analysis. Ppia , Hprt1 and Ywhaz were the most stable genes across the analysed brain areas and time points. B2m and Actb exhibited the greatest fluctuations, and the results for Gapdh were ambiguous.