Expression stability of six housekeeping genes: A proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR.
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.
Tibbi Biyoloji AD1, Mikrobiyoloji ve Klinik Mikrobiyoloji AD2 and Enfeksiyon Hastaliklari ve Klinik Mikrobiyoloji AD3, Kocaeli Universitesi, Tip Fakultesi, Sopali Ciftligi, 41900 Kocaeli, Turkey 4Genetik AD, DETAE, Istanbul Universitesi, Istanbul, Turkey.
Hakan
H
Savli
Tibbi Biyoloji AD1, Mikrobiyoloji ve Klinik Mikrobiyoloji AD2 and Enfeksiyon Hastaliklari ve Klinik Mikrobiyoloji AD3, Kocaeli Universitesi, Tip Fakultesi, Sopali Ciftligi, 41900 Kocaeli, Turkey 4Genetik AD, DETAE, Istanbul Universitesi, Istanbul, Turkey.
Aynur
A
Karadenizli
Tibbi Biyoloji AD1, Mikrobiyoloji ve Klinik Mikrobiyoloji AD2 and Enfeksiyon Hastaliklari ve Klinik Mikrobiyoloji AD3, Kocaeli Universitesi, Tip Fakultesi, Sopali Ciftligi, 41900 Kocaeli, Turkey 4Genetik AD, DETAE, Istanbul Universitesi, Istanbul, Turkey.
Fetiye
F
Kolayli
Tibbi Biyoloji AD1, Mikrobiyoloji ve Klinik Mikrobiyoloji AD2 and Enfeksiyon Hastaliklari ve Klinik Mikrobiyoloji AD3, Kocaeli Universitesi, Tip Fakultesi, Sopali Ciftligi, 41900 Kocaeli, Turkey 4Genetik AD, DETAE, Istanbul Universitesi, Istanbul, Turkey.
Sibel
S
Gundes
Tibbi Biyoloji AD1, Mikrobiyoloji ve Klinik Mikrobiyoloji AD2 and Enfeksiyon Hastaliklari ve Klinik Mikrobiyoloji AD3, Kocaeli Universitesi, Tip Fakultesi, Sopali Ciftligi, 41900 Kocaeli, Turkey 4Genetik AD, DETAE, Istanbul Universitesi, Istanbul, Turkey.
Ugur
U
Ozbek
Tibbi Biyoloji AD1, Mikrobiyoloji ve Klinik Mikrobiyoloji AD2 and Enfeksiyon Hastaliklari ve Klinik Mikrobiyoloji AD3, Kocaeli Universitesi, Tip Fakultesi, Sopali Ciftligi, 41900 Kocaeli, Turkey 4Genetik AD, DETAE, Istanbul Universitesi, Istanbul, Turkey.
Haluk
H
Vahaboglu
D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000235:genetics; D000890:Anti-Infective Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D002352:Carrier Proteins; D002939:Ciprofloxacin / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006602:Hexosyltransferases / Q000235:genetics; D006801:Humans; D008826:Microbial Sensitivity Tests; D009097:Multienzyme Complexes / Q000235:genetics; D002267:Muramoylpentapeptide Carboxypeptidase; D009154:Mutation; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D011762:Pyrroline Carboxylate Reductases / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D019105:Random Amplified Polymorphic DNA Technique; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012808:Sigma Factor / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics
D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D012333:RNA, Messenger; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; D002939:Ciprofloxacin; D011762:Pyrroline Carboxylate Reductases; D000217:Acyltransferases; D051080:Acyl-Carrier Protein S-Malonyltransferase; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D002267:Muramoylpentapeptide Carboxypeptidase; C048795:AmpC beta-lactamases; D001618:beta-Lactamases