, 2007). Transcriptional regulation of gene expression is crucial for progression of Chlamydia development. Furthermore, it is known that Chlamydia regulates transcription under stress conditions [e.g. caused by depletion of tryptophan, iron, arginine, or heat-shock Z-VAD-FMK datasheet response, which may restrain or block the developmental cycle (Wilson & Tan, 2002; Hogan et al., 2004; Ouellette et al., 2006; Schaumburg & Tan, 2006; Maurer et al., 2007)]. Chlamydia RNA

has previously been normalized against reference molecules such as 16S rRNA gyrA, and groEL_1 (Douglas & Hatch, 2000; Mathews et al., 2001; Belland et al., 2003; Nicholson et al., 2004; Goellner et al., 2006; Bailey et al., 2007; Kiselev et al., 2007; Maurer et al., 2007; Suchland et al., 2008; Klos et al., 2009). In addition, DNA has been used as an internal control (Ouellette et al., 2005, 2006; Carlson et al., 2008). Experiments performed by our group have emphasized the necessity of using appropriate controls to adequately address the expression of virulence-associated genes. Accordingly, the purpose of the present study was to compare and validate the use of RNA and DNA as internal gene expression controls during the early phase of the developmental cycle of Chlamydia pneumoniae. Our results suggest that, at least in the early phase of Chlamydia development,

Ixazomib mouse DNA is most suitable as an internal expression control due to its presence, stability, and correlation with bacterial proliferation. The chemical compound INP0010 was synthesized and purified from commercially available hydrazides and salicylaldehydes, as described previously (Kauppi et al., 2003; Nordfelth et al., 2005). INP0010 was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) immediately before each experiment. Rifampicin was dissolved in methanol and stored at −20 °C until use. Cells of the human epithelioid line HEp-2 (American Type Culture Collection, Rockville, MD; ATCC-CCL23) were grown in Roswell Park Memorial

Institute 1640 (Sigma-Aldrich) supplemented with 10% fetal calf serum (PromoCell), 20 mM HEPES (pH 8.0), 8 μg mL−1 garamycin (Schering–Plough), 1 μg mL−1 amphotericin B (Fungizone, Gibco), and l-glutamine (Sigma-Aldrich). The incubations were performed at 37 °C in the presence of 5% CO2 (Bailey et al., 2007). Testing with PLEK2 a mycoplasma detection kit (Stratagene, Cambridge, UK) indicated that the HEp-2 cells and bacteria were negative for mycoplasma infection. The C. pneumoniae strain T45 (kindly provided by J. Boman) was propagated in a HEp-2 cell infection system as described by Boman and colleagues (Kuoppa et al., 2002). HEp-2 cells were seeded in 6- or 24-well tissue culture plates. Chlamydia pneumoniae was added at a multiplicity of infection of 1 : 1 or 10 : 1 (used for RNA-stability measurements), and the plate was centrifuged at 1455 g for 1 h at 37 °C in a Sorvall RT 6000D.