p values less than 0.05 were considered a

significant difference. We thank Pamela Gardner for laboratory management. This study was supported by the Heart and Stroke Foundation of Canada (T6709, Zhang Z.-X.), the Canadian Institutes of Health Research (Zhang Z.-X.), and the Program of Experimental Medicine OTX015 (POEM) at University of Western Ontario London, Ontario, Canada (R3817001, Zhang Z.-X.). The authors declare no financial or commercial conflict of interest. “
“Many microbial pathogens invade and proliferate within host cells and the molecular mechanism underlying this behavior is currently being revealed for several bacterial species. Testing clinically relevant antibacterial compounds and elucidating their effects on gene expression requires adequate controls, especially when studying genetically intractable organisms such as Chlamydia spp., for which various gene fusions cannot be constructed. Apoptosis Compound Library clinical trial Until now, relative mRNA levels in Chlamydia have been measured using different internal gene expression controls, including 16S rRNA, mRNAs, and DNA. Here, we compared the advantages and disadvantages of various internal expression controls during the early phase of Chlamydia pneumoniae development. The relative abundance of target mRNAs varied

using the different internal control RNAs. This was partly due to variation in the transcript stability of the RNA species. Also, seven out of nine of the analyzed RNAs increased fivefold or more between 2 and Obeticholic Acid datasheet 14 h postinfection, while the amount of DNA and number of cells remained essentially unaltered. Our results suggest that RNA should not be used as a gene expression control during the

early phase of Chlamydia development, and that intrinsic bacterial DNA is preferable for that purpose because it is stable, abundant, and its relative amount is generally correlated with bacterial numbers. Members of the family Chlamydiaceae are strictly obligate intracellular bacteria that undergo a unique biphasic developmental cycle: first they exist as metabolically inert elementary bodies (EBs) that enter a host cell; after internalization, the EBs are enclosed in a membrane-bound compartment (the inclusion), where they differentiate into the metabolic and reproducing form called reticulate bodies (RBs). Inside the inclusion, the bacteria proliferate and escape the endocytic pathway of the host cell (Fields & Hackstadt, 2002). Gene expression patterns in Chlamydia are well-characterized, and thus transcriptional analysis has become a useful strategy to address experimentally challenging problems related to Chlamydia–host interactions (Fields et al., 2003; Slepenkin et al., 2003; Lugert et al., 2004). Unfortunately, no genetic tools are available that can determine the function of different genes in Chlamydia, although this problem might be circumvented using small inhibitory molecules (Peters et al., 2007).