[Real-time RT PCR with DNA subtraction for relative quantification of gene expression in Staphylococcus aureus].
OBJECTIVE
We evaluated relative quantification by real-time RT PCR of a target gene transcription.


METHODS
On the basis of (1+E)(-deltadeltaCt) 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 DNase I was not included in this protocol.


RESULTS
The gene expression of staphylococcal enterotoxin A (sea), 16S rRNA and RNA III of Staphylococcus aureus were detected. These two different analysis methods, DNA subtraction method and absolute quantitative method, led to similar results (p > 0.05).


CONCLUSIONS
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.
Xiang
Li
L
Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin 150030, China. xianglibio@163.com
Jiang
Yujun
Y
Liu
Wei
W
Bi
Yuhan
Y
Zhao
Feng
F
Huo
Guicheng
G
D002138:Calibration; D004269:DNA, Bacterial / Q000235:genetics*; D004768:Enterotoxins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013211:Staphylococcus aureus / Q000235:genetics*; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004768:Enterotoxins; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; C026066:enterotoxin A, Staphylococcal