Effects of aging, dietary restriction and glucocorticoid treatment on housekeeping gene expression in rat cortex and hippocampus-evaluation by real time RT-PCR.
Accurate normalization is the prerequisite for obtaining reliable results in the quantification of gene expression. Using TaqMan Real Time RT-PCR, we carried out an extensive evaluation of five most commonly used endogenous controls, gapdh, beta-actin, 18S rRNA, hprt and cypB, for their presumed stability of expression, in rat cortex and hippocampus, during aging, under dietary restriction and dexamethasone treatment. Valid reference genes (HKGs) were identified using GeNorm and NormFinder software packages and by direct comparison of Ct values. Analysis revealed gapdh and beta-actin as the most stable HKGs for all treatments analyzed, combined or separately, in the cortex, while in the hippocampus gapdh/hprt and beta-actin/hprt are the combination of choice for the single or combined effects of dietary restriction/dexamethasone, respectively. All treatments significantly influenced expression of 18S rRNA and cypB in both structures. In addition, we used gapdh and normalization factor, calculated by GeNorm, to compare the expression of alpha-syn in the cortex. Our results demonstrate the importance of the right choice of HKG and suggest the appropriate endogenous control to be used for TaqMan RT-PCR analysis of mRNA expression in rat cortex and hippocampus for selected experimental paradigms.
Tanic
Nikola
N
Institute for Biological Research, Bul. Despota Stefana 142, 11 060 Belgrade, Serbia.
Perovic
Milka
M
Mladenovic
Aleksandra
A
Ruzdijic
Sabera
S
Kanazir
Selma
S
D000199:Actins / Q000235:genetics; D000375:Aging / Q000502:physiology*; D000818:Animals; D031204:Caloric Restriction; D002540:Cerebral Cortex / Q000502:physiology*; D021983:Cyclophilins / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D020869:Gene Expression Profiling / Q000592:standards*; D005938:Glucocorticoids / Q000494:pharmacology*; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000235:genetics; D006624:Hippocampus / Q000502:physiology*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008297:Male; D019696:Peptidylprolyl Isomerase / Q000235:genetics; D011786:Quality Control; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000199:Actins; D005938:Glucocorticoids; D012337:RNA, Ribosomal, 18S; C081786:cyclophilin B; D003907:Dexamethasone; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D007041:Hypoxanthine Phosphoribosyltransferase; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase