Mastercycler r realplex2 real time pcr system

Gene expression analysis by RT-PCR was performed upon HOSCN treatment of PA14 wild-type and PA14 ppk strains. Briefly, cells were grown aerobically to mid-log phase in MOPS glucose at 37°C. HOSCN was added to a final concentration of 0.25 mM and incubated for 20 min. RNA was prepared using the NucleoSpin RNA kit (Macherey&Nagel) and DNA-free^™ kit (Ambion). PrimeScript 1^st strand cDNA Synthesis Kit (Takara) was used to generate cDNA, and RT-PCRs were set up with SYBR^(R) GreenER^™ qRT-PCR mix (Invitrogen) and a Mastercycler^(R) realplex2 real-time PCR system (Eppendorf). Expression ratios were calculated compared with expression of each gene in untreated PA14 and PA14 ppk cultures, respectively, by the ΔΔCT method (Pfaffl, 2001 (link)) and normalized to expression of rrsD (encoding 16S rRNA), expression of which did not change under the conditions tested. Primers used for RT-PCR analysis are listed in Table S3.

UTI89 wild-type and UTI89Δppk strains were cultivated and treated as described before prior to gene expression analysis by RT-PCR with the only modification that transcription was stopped with methanol after only 15 min of recovery at 37°C post treatment at 70°C for 1 min. RNA was prepared using the NucleoSpin RNA kit (Macherey & Nagel) and DNA-free™ kit (Ambion). PrimeScript 1^st strand cDNA Synthesis Kit (Takara) was used to generate cDNA, and RT-PCRs were set up with SYBR^(R) GreenER™ qRT-PCR mix (Invitrogen) and a Mastercycler^(R) realplex2 real-time PCR system (Eppendorf). Expression ratios were calculated compared with expression of each gene in untreated UTI89 and UTI89Δppk cultures, respectively, by the 2ΔΔCT method [37 (link)] and normalized to expression of rrsD (encoding 16S rRNA), expression of which did not change under the conditions tested. Primers used for RT- PCR analysis were: ibpAfw: 5’-ACAACCAGAGCCAGAGTAATGG-3’; ibpArev: 5’TATCCTGGGCGGTAATTTCC-3’; ibpBfw: 4’-AAAGCGACGATAACCACTACCG-3’; ibpBrev: 5’-CGTAAAGCTCAGGCTAAAAGGC-3’; rrsDfw 5′-AAG AAC TTA CCT GGT CTT GAC ATC-3′; rrsDrev 5′-CAG TTT ATC ACT GGC AGT CTC CTT-3′.