Selection and evaluation of stable housekeeping genes for gene expression normalization in carbon nanoparticle-induced acute pulmonary inflammation in mice Renfu Yin a,c,d , Furong Tian a , Birgit Frankenberger a , Martin Hrabé de Angelis b,c , Tobias Stoeger a,⇑ a Comprehensive Pneumology Center, Institute of Lung Biology and Disease (iLBD), Helmholtz Zentrum München, Ingolstädter Landstraße 1, D-85764 Neuherberg/Munich, Germany b Institute of Experimental Genetics, Helmholtz Zentrum München, Ingolstädter Landstraße 1, D-85764 Neuherberg/Munich, Germany c Lehrstuhl für Experimentelle Genetik, Technische Universität München, Freising Weihenstephan 85354, Germany d Department of Veterinary Preventive Medicine, College of Animal Science and Veterinary Medicine, Jilin University, Xi’an Road 5333, Changchun, Jilin 130062, China article info Article history: Received 16 July 2010 Available online 1 August 2010 Keywords: Housekeeping genes Acute lung inflammation NF-jB Quantitative RT-PCR abstract Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a highly specific and sensitive technique for the quantification of gene expression on the mRNA levels. But use of unconfirmed house- keeping genes (HKGs) could lead to misinterpretation of the expression of genes of interest (GOI). In this study, the stability and suitability of 11 frequently used housekeeping genes, namely 18S rRNA, ACTB, B2M, CYPA, GADPH, GUSB, HMBS, HPRT1, RPL13A, SDHA and TBP in 36 lung tissues isolated from either wild-type (WT) mice or p50 knock out (p50À/À) mice or p105 knock-out (p105À/À) mice which were treated with either carbon nanoparticle (CNP) or H 2 O or non-treated, have been validated by geNorm, NormFinder and BestKeeper programs. The expression levels of ACTB, GUSB and RPL13A were the most constant in lung tissues across three genotypes and three kinds of treatments. A set of three most stable genes is found sufficient to be used as housekeeping genes for lung tissues in studies of similar design. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Inhalation of carbon nanoparticles (CNP), a main constituent of urban air pollution, is believed to trigger pulmonary or even sys- temic inflammation via the generation of oxidative stress [1,2]. The redox-sensitive transcription factor NF-jB, which controls a majority of inflammatory genes, is thought to play an important role in onset of pulmonary inflammation [3,4]. In mammalian cells, the NF-jB family is composed of five members, NF-jB1 (p50, pre- cursor p105), NF-jB2 (p52, precursor p100), RelA, RelB and c-Rel, which function as various hetero- and homo-dimmers [5]. It has been reported that NF-jB1 (p50 and p105) plays import roles in NF-jB functions, however, whether the subunit p50 and p105 of NF-jB could control acute pulmonary inflammation and injury after 24 h upon CNP treatment is not clear. One approach to understanding p50 and p105 roles in CNP-in- duced acute pulmonary inflammation is to study gene expression in animal models using qRT-PCR. The data obtained by qRT-PCR are typically normalized with an internal control, often referred to as a housekeeping gene. However, the use of unconfirmed HKGs may lead to misinterpretation of the expression of GOI. Up to now, several mathematical methods, such as geNorm [6], NormFinder [7] and BestKeeper [8], have been developed to analyze the vari- ability of the expression of candidate HKG. The ideal HKG for qRT-PCR would be one whose mRNA is consistently expressed at the same level in all samples under investigation, regardless of tis- sue type, disease state, medication or experimental conditions, and could have expression levels comparable to that of the target [9– 11]. However, a systematic study of the suitability of HKGs for qRT- PCR normalization in the field of CNP-induced acute pulmonary inflammation has thus far been lacking. Therefore, the aim of the present study is to identify candidate genes in the CNP-induced acute pulmonary inflammation models that could be used in qRT-PCR experiments as housekeeping genes to normalize the expression of GOI. 2. Methods 2.1. Animal treatment and lung tissue processing Animal treatment and lung tissue processing as described in our previous studies [1,35]. Briefly, all mice were female, 10–12 weeks of age with body weights between 17.39 and 20.5 g during the study. Each of three genetically modified mice consisted of three 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.07.104 ⇑ Corresponding author at: Institute of Lung Biology and Disease (iLBD), Helmholtz Zentrum München, Ingolstädter Landstrasße 1, D-85764 Neuherberg/ Munich, Germany. Fax: +49 (0) 89 3187 2400. E-mail address: tobias.stoeger@helmholtz-muenchen.de (T. Stoeger). Biochemical and Biophysical Research Communications 399 (2010) 531–536 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc