Abstract
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a very sensitive widespread technique considered as the gold standard to explore transcriptional variations. While a particular methodology has to be followed to provide accurate results many published studies are likely to misinterpret results due to lack of minimal quality requirements. Yersinia pestis is a highly pathogenic bacterium responsible for plague. It has been used to propose a ready-to-use and complete approach to mitigate the risk of technical biases in transcriptomic studies. The selection of suitable reference genes (RGs) among 29 candidates was performed using four different methods (GeNorm, NormFinder, BestKeeper and the Delta-Ct method). An overall comprehensive ranking revealed that 12 following candidate RGs are suitable for accurate normalization: gmk, proC, fabD, rpoD, nadB, rho, thrA, ribD, mutL, rpoB, adk and tmk. Some frequently used genes like 16S RNA had even been found as unsuitable to study Y. pestis. This methodology allowed us to demonstrate, under different temperatures and states of growth, significant transcriptional changes of six efflux pumps genes involved in physiological aspects as antimicrobial resistance or virulence. Previous transcriptomic studies done under comparable conditions had not been able to highlight these transcriptional modifications. These results highlight the importance of validating RGs prior to the normalization of transcriptional expression levels of targeted genes. This accurate methodology can be extended to any gene of interest in Y. pestis. More generally, the same workflow can be applied to identify and validate appropriate RGs in other bacteria to study transcriptional variations.
Highlights
Plague is a fatal disease caused by the Gram-negative bacterium Yersinia pestis[1] which has been responsible for approximately 200 million deaths in the past and is still a global public health issue
A total of 29 candidate reference genes (RGs) were selected (Table 1) on the basis of our laboratory experience and of an extended literature screening of genes designated as “housekeeping” gene in Yersinia genus[28,29] and other genes commonly used as RGs in other bacteria species and present in Y. pestis genome[24,25,26,30]
The quantification cycle (Cq) values from each of the 29 candidate RGs were generated by RT-qPCR using RNA extracted from exponential and stationary-phase of Y. pestis grown at 21 °C, 26 °C and 37 °C in Luria-Bertani (LB) broth
Summary
We found that gmk is the most stably expressed gene followed by proC, fabD, rpoD, nadB, rho, thrA, ribD, mutL rpoB, adk and tmk Those twelve genes were considered stable enough to be employed as RGs by the four previously detailed methods and were the best candidates for RT-qPCR experiment in Y. pestis. Based on the expression of five ranked most stable genes gmk, proC, fabD, rpoD and nadB given by RefFinder according to the GeNorm method, the pairwise variation (Vn/Vn+1) was calculated between the normalization factors NFn and NFn+1 to determine the optimal number of required RGs. The genes inclusion has been stopped when the pairwise variation reached the cut-off value below 0.15, in our case after the fourth ranked gene. Three differences have been found as statistically significant with the 16S RNA normalization, but not with our RGs
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