RT-PCR and qRT-PCR analyses of the hasA upstream region.

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RT-PCR and qRT-PCR analyses of the hasA upstream region. RT-PCR and qRT-PCR analyses of the hasA upstream region. (A) Schematic representation of the hasA upstream region. Shown is the approximate location of the promoters (bent arrows). Horizontal arrows indicate the positions of the primers used for RT-PCR. (B) RT-PCR confirming the transcription of the hasA upstream region. MGAS2221 gDNA and cDNA from reaction mixtures containing (+RT) or not containing (−RT) reverse transcriptase were used as the templates in four PCRs. The relative locations of the primers used (numbered R, F1, F2, F3, and F4) are shown in panel A. The samples without reverse transcriptase served as a control against gDNA contamination. (C) qRT-PCR analysis of the read-through transcription. Graph showing the mean values of fold change for the read-through mRNA levels in the mutants relative to those of MGAS2221. Both WT and mutant values were relative to those of the internal control gene, plr, with mutant values representing the fold change relative to that of WT, which was converted to 1. The experiments were performed in triplicate, and mean values ± SDs are shown. * indicates statistically significant differences relative to WT (P < 0.05, unpaired t test). Marina Falaleeva et al. Infect. Immun. 2014;82:5293-5307