Identifying stable reference genes for evaluation of antioxidative enzyme gene expression in auditory cortex and cochlea of young and old Fischer 344 rats.
CONCLUSIONS
This study suggests that the validation of reference genes is important for the accurate evaluation of differential gene expression in age-related hearing loss (AHL).


OBJECTIVE
To identify the most stable reference gene in the auditory cortex and cochlea of young and old rats. Materials and methods. mRNA levels of some common reference genes such as beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C, hypoxanthine phosphoribosyl-transferase and eukaryotic translation elongation factor (EF) were quantified in the auditory cortex and cochlea of young and old rats by quantitative RT-PCR, and then their stability was evaluated using GeNorm and Normfinder software.


RESULTS
There were some variations in the expression of reference genes found in the auditory cortex and cochlea of rats, wherein EF and GAPDH were identified as the most stable reference genes in auditory cortex and cochlea, respectively. mRNA levels of Cu/Zn-superoxide dismutase (SOD), Mn-SOD, catalase and glutathione peroxidase in the auditory cortex and cochlea do not vary significantly in young and old rats, when they are normalized with EF or GAPDH, the valid reference genes, respectively. However, Cu/Zn-SOD and catalase levels differ significantly in the auditory cortex when they are normalized with beta-actin, an invalid reference gene, resulting in the misinterpretation of the target gene expression levels.
Chen
Jie
J
Cancer and Ageing Research Group, Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos #04-01, Singapore.
Ruan
Runsheng
R
D000199:Actins / Q000235:genetics; D000375:Aging / Q000235:genetics*; D000818:Animals; D001303:Auditory Cortex / Q000201:enzymology*; D002374:Catalase / Q000235:genetics*; D003051:Cochlea / Q000201:enzymology*; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D034381:Hearing Loss / Q000201:enzymology*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008297:Male; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D013482:Superoxide Dismutase / Q000235:genetics*; D025822:Ubiquitin C / Q000235:genetics
D000199:Actins; C094814:EEF1A1 protein, human; D020648:Peptide Elongation Factor 1; D012333:RNA, Messenger; D025822:Ubiquitin C; D002374:Catalase; D013482:Superoxide Dismutase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007041:Hypoxanthine Phosphoribosyltransferase