Validation of suitable internal control genes for expression studies in aging.
Quantitative data from experiments of gene expression are often normalized through levels of housekeeping genes transcription by assuming that expression of these genes is highly uniform. This practice is being questioned as it becomes increasingly clear that the level of housekeeping genes expression may vary considerably in certain biological samples. To date, the validation of reference genes in aging has received little attention and suitable reference genes have not yet been defined. Our aim was to evaluate the expression stability of frequently used reference genes in human peripheral blood mononuclear cells with respect to aging. Using quantitative RT-PCR, we carried out an extensive evaluation of five housekeeping genes, i.e. 18s rRNA, ACTB, GAPDH, HPRT1 and GUSB, for stability of expression in samples from donors in the age range 35-74 years. The consistency in the expression stability was quantified on the basis of the coefficient of variation and two algorithms termed geNorm and NormFinder. Our results indicated GUSB be the most suitable transcript and 18s the least for accurate normalization in PBMCs. We also demonstrated that aging is a confounding factor with respect to stability of 18s, HPRT1 and ACTB expression, which were particularly prone to variability in aged donors.
Zampieri
M
M
Department of Cellular Biotechnologies and Hematology, II Faculty of Medicine and Surgery, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy.
Ciccarone
F
F
Guastafierro
T
T
Bacalini
M G
MG
Calabrese
R
R
Moreno-Villanueva
M
M
Reale
A
A
Chevanne
M
M
Bürkle
A
A
Caiafa
P
P
D000328:Adult; D000368:Aged; D000375:Aging / Q000235:genetics*; D000465:Algorithms; D015870:Gene Expression; D005796:Genes; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism / Q000592:standards*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008875:Middle Aged; D012333:RNA, Messenger / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase