Computational protocol: A gacS Deletion in Pseudomonas aeruginosa Cystic Fibrosis Isolate CHA Shapes Its Virulence

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Protocol publication

[…] The strains were grown at 37°C under agitation in LB, that was supplemented with 5 mM EGTA and 20 mM MgCl2 (conditions of in vitro T3SS induction) for Reverse Transcriptase (RT)-qPCR analysis. Total RNA was either extracted with the TRIzol Plus RNA Purification Kit (Invitrogen) then treated with DNase I (Amplification Grade, Invitrogen), or the PureYield RNA Midiprep System (Promega), cleaned up and concentrated using the RNeasy kit (Qiagen). Yield, purity and integrity of RNA were further evaluated on Nanodrop and by agarose gel migration. Complementary DNA synthesis was carried out with SuperScript III First-Strand Synthesis System (Invitrogen) in presence or not of the SuperScript III RT enzyme to assess the absence of genomic DNA. For RT-PCR experiments, performed to assess the presence of GacS mRNA, PCR amplifications were performed using TaqPCRx DNA Polymerase (Invitrogen) and following the Basic PCR protocol described by the manufacturer. Calibration of the PCR amplification steps was done by varying number of cycles with primers targeting either gacS or 16S rRNA as a reference transcript. The RT-qPCR runs, used to quantify the effect of GacS on its target genes, were carried out on a CFX96 Real-Time System (Bio-Rad). Cycling parameters of the real time PCR were 98 °C for 2 min, following by 45 cycles of 98°C for 5 s and 60°C for 10 s, ending with a melting curve from 65 °C to 95 °C to assess the specificity of the amplification. To determine the amplification kinetics of each product, the fluorescence derived from the incorporation of EvaGreen into the double-stranded PCR products was measured at the end of each cycle using the SsoFast EvaGreen Supermix 2X Kit (Biorad). The results were analyzed using the Bio-Rad CFX Manager Software 3.0 (Bio-Rad). The relative mRNA quantity of each gene under GacS production as compared to absence of production was analyzed using the Relative Expression Software Tool REST2009 (Qiagen) with a pair wise fixed reallocation randomization test coupled to a standard error (SE) calculated via a Taylor algorithm. The 16S rRNA was used as reference for normalization. The sequences of all primers are given in . […]

Pipeline specifications

Software tools CFX Manager, REST
Application qPCR
Organisms Pseudomonas aeruginosa, Homo sapiens
Diseases Cystic Fibrosis, Infection