How to validate the RT-qPCR method in SybrGreen detection? Application to the analysis of the transcriptomic response of Campylobacter jejuni under stress

Abstract

The transcriptional analysis via the use of the RT-qPCR technique (Reverse Transcription coupled with quantitative PCR) is largely used even though the growing use of high flow sequencing techniques. The RT-qPCR allows to quantify the level of mensenger ARN of genes reflecting the level of expression of these genes. It has numerous applications that require a high caution when implementing it. This technique needs the validation of the different steps of the procedure and numerous tests need to be done before the experimentation as well as controls during the experimentation. The objective of this paper is to describe the different control points and to detail vigilance points to be respected during the use of the RT-qPCR. From the extraction of nuclear acids to final results analysis, the validation of the different steps has demonstrated a reliable and robust method for the publication of works about the transcriptomic response of bacterian pathogene Campylobacter jejuni submitted to thermal stress.