139 Molecular and Cellular Biochemistry 236: 139–146, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands. Differential expression of GAPDH and β -actin in growing collateral arteries Elisabeth Deindl, Kerstin Boengler, Niels van Royen and Wolfgang Schaper Max-Planck-Institute, Department of Experimental Cardiology, Bad Nauheim, Germany Received 8 June 2001; accepted 1 March 2002 Abstract Housekeeping genes like glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are often used as internal stand- ards for quantitative RNA analysis. In our study we analyzed the relative expression level of GAPDH and β-actin as well as of the 18S rRNA and the Poly (A) + RNA in growing collateral arteries in a rabbit model of arteriogenesis which is not associated with ischemia. Relative quantitation of the housekeeping genes displayed a significant upregulation of the β-actin- and GAPDH mRNA during the first 24 h of vessel growth. For day 3 our results revealed an even stronger upregulation of the β-actin mRNA (140%) but a significant downregulation of the GAPDH mRNA (50% of control). The 18S rRNA, however, showed for the same periods only minor alterations compared to the Poly (A) + RNA. From these results we conclude that the 18S rRNA, but not the GAPDH- or β-actin mRNA is an appropriate internal control for relative quantitation of gene expression under condi- tions of cell proliferation in growing vessels. (Mol Cell Biochem 236: 139–146, 2002) Key words: GAPDH, β-actin, 18S rRNA, Poly (A) + RNA, relative quantitation of gene expression, collateral artery growth Introduction Monitoring gene expression became an important component in many cardiovascular research projects. Numerous options for the quantitation of mRNA expression such as Northern blot, nuclease protection assay or RT-PCR are available to the researcher today. Relative quantitation compares transcript abundance across multiple samples using an endogenous internal control for sample normalization. Ideally, an inter- nal control will not show variation in expression across ex- perimental samples. Matched loading based on an internal control is critical for quantitative comparison of gene expres- sion among different tissue types, varying developmental stages and experimental treated cells. Constitutively ex- pressed housekeeping genes such as GAPDH or β-actin are often used as internal controls. Unfortunately, these genes are not always expressed at constant levels under the different experimental conditions [1–9]. Therefore, it is necessary to characterize the suitability of various genes to serve as in- ternal RNA controls under particular experimental conditions where differential gene expression is investigated. Expression profiling in growing vessels became of increas- ing interest in the different fields of cardiovascular and can- cer research. Many studies investigating gene expression during the process of capillary sprouting (angiogenesis) or collateral artery growth (arteriogenesis) have been performed using e.g. GADPH or β-actin as internal control [10, 11]. However, both types of vessel growth do not obey the same molecular mechanisms [12–14]. Capillary sprouting is mainly observed in ischemic territories [15–17]. Collateral artery growth, however, is often remote from ischemia and tis- sue ischemia seems not to play a causative role for arterio- genesis [16, 18]. The present study was designed to evaluate GAPDH, β- actin and the 18S rRNA for their suitability to serve as inter- nal controls for expression profiling in growing collateral Address for offprints: E. Deindl, Max-Planck-Institute, Department of Experimental Cardiology, Benekestrasse 2, D-61231 Bad Nauheim, Germany (E-mail: e.deindl@kerckhoff.mpg.de)