## Validation of suitable internal control genes for expression studies in aging

2010
##### Authors
Zampieri, Michele
Ciccarone, Fabio
Guastafierro, Tiziana
Bacalini, Maria Giulia
Calabrese, Roberta
Reale, Anna
Chevanne, Marta
Caiafa, Paola
200880
Journal article
##### Published in
Mechanisms of Ageing and Development ; 131 (2010), 2. - pp. 89-95. - ISSN 0047-6374. - eISSN 1872-6216
##### Abstract
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.
##### Subject (DDC)
570 Biosciences, Biology
##### Keywords
Aging,Housekeeping genes,GUSB,Real-time PCR normalization,PBMC
##### Cite This
ISO 690ZAMPIERI, Michele, Fabio CICCARONE, Tiziana GUASTAFIERRO, Maria Giulia BACALINI, Roberta CALABRESE, Maria MORENO-VILLANUEVA, Anna REALE, Marta CHEVANNE, Alexander BÜRKLE, Paola CAIAFA, 2010. Validation of suitable internal control genes for expression studies in aging. In: Mechanisms of Ageing and Development. 131(2), pp. 89-95. ISSN 0047-6374. eISSN 1872-6216. Available under: doi: 10.1016/j.mad.2009.12.005
BibTex
@article{Zampieri2010Valid-7866,
year={2010},
title={Validation of suitable internal control genes for expression studies in aging},
number={2},
volume={131},
issn={0047-6374},
journal={Mechanisms of Ageing and Development},
pages={89--95},
author={Zampieri, Michele and Ciccarone, Fabio and Guastafierro, Tiziana and Bacalini, Maria Giulia and Calabrese, Roberta and Moreno-Villanueva, Maria and Reale, Anna and Chevanne, Marta and Bürkle, Alexander and Caiafa, Paola}
}

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<dcterms:abstract xml:lang="eng">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.</dcterms:abstract>
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