Expression stability of six housekeeping genes:: a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR

被引:263
作者
Savli, H
Karadenizli, A
Kolayli, F
Gundes, S
Ozbek, U
Vahaboglu, H
机构
[1] Kocaeli Univ, Tip Fak, Tibbi Biyoloji AD, TR-41900 Kocaeli, Turkey
[2] Kocaeli Univ, Tip Fak, Mikrobiyol Ve Mikrobiyol AD, TR-41900 Kocaeli, Turkey
[3] Kocaeli Univ, Tip Fak, Enfeksiyon Hastaliklari Ve Klin Mikrobiyol AD, TR-41900 Kocaeli, Turkey
[4] Istanbul Univ, DETAE, Genet AD, Istanbul, Turkey
关键词
D O I
10.1099/jmm.0.05132-0
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 ends further support to the use of proC and rpoD genes simultaneously as internal controls for such studies.
引用
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页码:403 / 408
页数:6
相关论文
共 23 条
[1]   Correlation between protein and mRNA abundance in yeast [J].
Gygi, SP ;
Rochon, Y ;
Franza, BR ;
Aebersold, R .
MOLECULAR AND CELLULAR BIOLOGY, 1999, 19 (03) :1720-1730
[2]   AMPD, ESSENTIAL FOR BOTH BETA-LACTAMASE REGULATION AND CELL-WALL RECYCLING, IS A NOVEL CYTOSOLIC N-ACETYLMURAMYL-L-ALANINE AMIDASE [J].
JACOBS, C ;
JORIS, B ;
JAMIN, M ;
KLARSOV, K ;
VANBEEUMEN, J ;
MENGINLECREULX, D ;
VANHEIJENOORT, J ;
PARK, JT ;
NORMARK, S ;
FRERE, JM .
MOLECULAR MICROBIOLOGY, 1995, 15 (03) :553-559
[3]   Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction [J].
Johnson, MR ;
Wang, KS ;
Smith, JB ;
Heslin, MJ ;
Diasio, RB .
ANALYTICAL BIOCHEMISTRY, 2000, 278 (02) :175-184
[4]   INVESTIGATION OF A NOSOCOMIAL OUTBREAK OF PSEUDOMONAS-AERUGINOSA - PNEUMONIA IN AN INTENSIVE-CARE UNIT BY RANDOM AMPLIFICATION OF POLYMORPHIC DNA ASSAY [J].
KERR, JR ;
MOORE, JE ;
CURRAN, MD ;
GRAHAM, R ;
WEBB, CH ;
LOWRY, KG ;
MURPHY, PG ;
WILSON, TS ;
FERGUSON, WP .
JOURNAL OF HOSPITAL INFECTION, 1995, 30 (02) :125-131
[5]   Overexpression of the MexEF-OprN multidrug efflux system affects cell-to-cell signaling in Pseudomonas aeruginosa [J].
Köhler, T ;
van Delden, C ;
Curty, LK ;
Hamzehpour, MM ;
Pechere, JC .
JOURNAL OF BACTERIOLOGY, 2001, 183 (18) :5213-5222
[6]   Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster:: Purification of acyl carrier protein (ACP) and malonyl-coenzyme A:ACP transacylase (FabD) [J].
Kutchma, AJ ;
Hoang, TT ;
Schweizer, HP .
JOURNAL OF BACTERIOLOGY, 1999, 181 (17) :5498-5504
[8]   Assignment of the substrate-selective subunits of the MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa [J].
Maseda, H ;
Yoneyama, H ;
Nakae, T .
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 2000, 44 (03) :658-664
[9]   Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux pumps in Pseudomonas aeruginosa [J].
Masuda, N ;
Sakagawa, E ;
Ohya, S ;
Gotoh, N ;
Tsujimoto, H ;
Nishino, T .
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 2000, 44 (12) :3322-3327
[10]   Construction of a series of mutants lacking all of the four major mex operons for multidrug efflux pumps or possessing each one of the operons from Pseudomonas aeruginosa PAO1:: MexCD-OprJ is an inducible pump [J].
Morita, Y ;
Komori, Y ;
Mima, T ;
Kuroda, T ;
Mizushima, T ;
Tsuchiya, T .
FEMS MICROBIOLOGY LETTERS, 2001, 202 (01) :139-143