SHORT COMMUNICATION Validation of an internal control gene to apply reverse transcription quantitative PCR to study heat, cold and ethanol stresses in Lactobacillus plantarum D. Fiocco Æ E. Crisetti Æ V. Capozzi Æ G. Spano Received: 1 August 2007 / Accepted: 27 August 2007 / Published online: 20 September 2007 Ó Springer Science+Business Media B.V. 2007 Abstract The dissection of the stress tolerance in lactic acid bacteria (LAB) may improve our knowledge on the adaptive physiology of LAB and may facilitate the selec- tion of species suitable for industrial application. In this work we report the validation of the ldhD gene as internal control for reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in a model LAB such as Lactobacillus plantarum. The expression of the ldhD gene was analysed in lag phase, early, mid and late exponential phase and early and late stationary phase. Moreover, dif- ferent stress conditions were analysed: heat shock (40°C), cold shock (10°C) and ethanolic shock (10% v/v). The quantification of transcript levels of a small heat shock gene (hsp 18.55) after heat, cold and ethanol shock allowed us to validate this method. RT-qPCR appeared as a pow- erful tool to study L. plantarum response in stress conditions and the internal control used in this study may be extended to other species of LAB. Keywords RT-qPCR Á Lactobacillus plantarum Á Small heat shock genes Á ldhD Introduction Current methods used to study gene expression, such as Northern hybridization, quantitative competitive polymerase chain reaction (RT-qPCR), and RNase protection assays, have a small dynamic range and a lack of sensitivity (Vandecasteele et al. 2001). The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful technique, which allows the quantification of steady-state mRNA levels in bacteria (Corbella and Puyet 2003; Vandecasteele et al. 2003; Devers et al. 2004; Desr- oche et al. 2005). However, the real time PCR requires the use of internal controls (usually known as housekeeping) for data normalization. The expression of these genes is often considered to fluctuate very little in comparison to other genes or the genes that are analysed under determinate conditions, although in given conditions, their expression can vary considerably (Thellin et al. 1999; Vandecasteele et al. 2001; Desroche et al. 2005). The most frequent strat- egy implies the determination of an internal control gene to normalize mRNA fractions for each study. In this study we presented the validation of a housekeeping gene suitable for the analysis of gene expression during abiotic stresses in Lactobacillus plantarum, a versatile bacterium that is found in a variety of ecological niches, ranging from vegetable and plant fermentations to the human gastrointestinal tract (Ercolini et al. 2003; Aymerich et al. 2003; Spano et al. 2004a; Spano et al. 2006a). Moreover, L. plantarum is extensively used as model microorganism among Lactoba- cillus spp. (Spano et al. 2006b; de Vries et al. 2006). Recently several presumed housekeeping genes were used in order to analyse the stress response in the wine bacterium Oenococcus oeni using a real time PCR approach (Desroche et al. 2005). Among these, the ldhD gene, encoding D-lac- tate dehydrogenase, proved to be the best candidate as internal control for the analysis of gene expression during abiotic stresses in O. oeni (Desroche et al. 2005). The aim of this work was to analyse the potentiality of the ldhD gene as an internal control in L. plantarum. The D. Fiocco Á E. Crisetti Á V. Capozzi Á G. Spano (&) Department of Food Science, Foggia University, via Napoli 25, 71100 Foggia, Italy e-mail: g.spano@unifg.it Present Address: G. Spano BioAgromed, Foggia University, 71100 Foggia, Italy 123 World J Microbiol Biotechnol (2008) 24:899–902 DOI 10.1007/s11274-007-9556-7