Real-time RT PCR with DNA subtraction for relative quantification of gene expression in Staphylococcus aureus

Abstract:

[Objective] We evaluated relative quantification by real-time RT PCR of a target gene transcription. [Methods] On the basis of (1+E)-△△Ct mathematical model and the E=10[-1/slope]-1 equation, the detected Ct data of the real-time RT PCR was analyzed by the new DNA subtraction assay. DNA was used as standard for the initial amount of bacteria. RT and RT— samples for real-time PCR detection were prepared to quantify the DNA that simultaneously existed with RNA isolated from the bacteria samples. The detected quantitative data were subtracted from total nucleic acid simultaneously contained RNA and DNA. Enzymatic digestion with DNaseⅠwas not included in this protocol. [Results] The gene expression of staphylococcal enterotoxin A (sea), 16S rRNA and RNAⅢof Staphylococcus aureus were detected. These two different analysis methods, DNA subtraction method and absolute quantitative method, led to similar results (p>0.05). [Conclusion] This is a time-saving and efficient method. Additionally, for further studies it would be conceivable to extend the detection of genes expression from S. aureus to other prokaryote.