Use of gDNA as internal standard for gene expression in staphylococci in vitro and in vivo.
An internal RNA standard proved less suitable in bacterial gene expression experiments. We therefore developed a method for simultaneous RNA and gDNA (genomic DNA) isolation from in vitro and in vivo samples containing staphylococci and combined it with quantitative PCR. The reliability of gDNA for bacterial quantification and for standardisation in gene expression experiments was evaluated. Quantitative PCR proves equivalent to quantitative culture for in vitro samples, and superior for in vivo samples. In gene expression experiments, gDNA permits a good standardisation for the initial amount of bacteria. The average interassay variability of the in vitro expression is 20.1%. The in vivo intersample variability was 73.3%. This higher variability can be attributed to the biological variation of gene expression in vivo. This method permits exact quantification of the number of bacteria and the expression of genes in staphylococci in vivo (e.g., in biofilms, evolution in time) and in vitro.
Vandecasteele
S J
SJ
Infectious Diseases Research Group, Rega Institute for Medical Research, Leuven, Belgium. stefaan.vandecasteele@uz.kuleuven.ac.be
Peetermans
W E
WE
Merckx
R
R
Van Ranst
M
M
Van Eldere
J
J
D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D016680:Genome, Bacterial; D007700:Kinetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000302:isolation & purification; D012015:Reference Standards; D015203:Reproducibility of Results; D013210:Staphylococcus / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D013212:Staphylococcus epidermidis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D012329:RNA, Bacterial