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Expression stability of six housekeeping genes: a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR
- Source :
- Journal of Medical Microbiology. 52:403-408
- Publication Year :
- 2003
- Publisher :
- Microbiology Society, 2003.
-
Abstract
- Constantly expressed genes are used as internal controls in relative quantification studies. Suitable internal controls for such studies have not yet been defined for Pseudomonas aeruginosa. In this study, the genes ampC, fabD, proC, pbp-2, rpoD and rpoS of P. aeruginosa were compared in terms of expression stability by real-time quantitative RT-PCR. A total of 23 strains with diverse resistance phenotypes were studied. Stability of expression among the housekeeping genes was assessed on the basis of correlation coefficients, with the best-correlated pair accepted as being the most stable one. Eventually, proC and rpoD formed the most stable pair (r = 0.958; P < 0.001). Next, in four ciprofloxacin-selected nfxC-like mutants, levels of oprD, oprM and oprN mRNA were compared with those of their wild-type counterparts. The comparison was made after correcting the raw values by the geometric mean of the internal control genes proC and rpoD. The level of oprN mRNA was significantly up-regulated, while the oprD gene was down-regulated (although this difference was statistically insignificant), in the mutants. This expression pattern was consistent with that of the expected expression profile of nfxC-type mutants; this experiment therefore lends further support to the use of proC and rpoD genes simultaneously as internal controls for such studies.
- Subjects :
- DNA, Bacterial
Microbiology (medical)
Mutant
Sigma Factor
Microbial Sensitivity Tests
Muramoylpentapeptide Carboxypeptidase
Biology
Microbiology
beta-Lactamases
Anti-Infective Agents
Bacterial Proteins
Ciprofloxacin
Multienzyme Complexes
Drug Resistance, Bacterial
Gene expression
Acyl-Carrier Protein S-Malonyltransferase
Humans
Penicillin-Binding Proteins
RNA, Messenger
Gene
DNA Primers
Genetics
Reverse Transcriptase Polymerase Chain Reaction
DNA-Directed RNA Polymerases
Gene Expression Regulation, Bacterial
General Medicine
Phenotype
Random Amplified Polymorphic DNA Technique
Housekeeping gene
Reverse transcription polymerase chain reaction
Real-time polymerase chain reaction
Hexosyltransferases
Mutation
Peptidyl Transferases
Pseudomonas aeruginosa
Pyrroline Carboxylate Reductases
Carrier Proteins
rpoS
Acyltransferases
Subjects
Details
- ISSN :
- 14735644 and 00222615
- Volume :
- 52
- Database :
- OpenAIRE
- Journal :
- Journal of Medical Microbiology
- Accession number :
- edsair.doi.dedup.....efe56212125d792e4c170cbd17f66b0c