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. 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. Between 2010 and 2015, the World Health Organization (WHO) reported more than 3200 cases with more than 500 deaths 2. In 2017, at least 64 patients succumbed during an outbreak in Madagascar 3. Plague mostly circulates in rodent populations through the bites of infected fleas. Bubonic plague is the usual presentation in humans after fleabites while primary septicemia without detectable bubos is possible. Pneumonic plague may occur after bubonic and septicemic forms. At this stage, bacteria are transmissible by air, causing deadly primary pneumonic presentations 1. Thus, life cycle of Y. pestis between the mammalian hosts through the flea vectors implies that the bacterium senses the oscillations of its environmental temperature. Therefore, it is not surprising that temperature controls several biological processes playing a key role in the colonization of both the mammalian host and the flea vector 4. In fact, previous comparative transcriptomic and proteomic studies have shown that many genes are differentially expressed when temperature increases from 21/28 to 37 °C and inversely 5. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widespread 6 gold standard 7 technique to explore transcriptional variations. It is often the easiest and the most cost effective solution