Gene Expression Analysis in Bacteria by RT-qPCR

• DOI: 10.1007/978-1-4939-9833-3_10
• Publication Date: 2019-10-02T14:06:24Z
• Authors: Danilo J. P. G. R...; Thiago L. P. Castro; Eric R. G. R. Aguiar; Luis G. C. Pacheco
• Keywords: 0301 basic medicine; 0303 health sciences; Genes, Essential; Bacteria; Gene Expression Profiling; Molecular Probe Techniques; Reproducibility of Results; Guidelines as Topic; Gene Expression Regulation, Bacterial; Reference Standards; Real-Time Polymerase Chain Reaction; 03 medical and health sciences; RNA, Bacterial; Molecular Probes; Fluorescent Dyes

Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) using fluorescent DNA-binding dyes is now a gold-standard methodology to study bacterial gene expression through relative quantitation of target mRNAs under specific experimental conditions, and recent developments in the technology allow for gene expression analysis in single cells. Nevertheless, several critical steps of the RT-qPCR protocol need to be carefully addressed in order to obtain reliable results, particularly regarding RNA sample quality and appropriate choice of reference genes. Besides, accurate reporting of study conditions is essential, as recommended by the MIQE guidelines. Herein, we provide a practical approach to quantitation of the transcript levels of bacterial genes using RT-qPCR, including a general protocol for obtaining good-quality bacterial RNA and a discussion on the selection and validation of candidate bacterial reference genes for data normalization.