FIG. 2.

Detection and quantification of iupA mRNA. (A) RT-PCR of iupA mRNA (top panel) and 16S rRNA (bottom panel) from two independent experiments. Lane 1, 100-bp size marker; lanes 2 and 3, RNA isolated from R. equi grown under iron-replete conditions; lanes 4 and 5, RNA isolated from R. equi grown under iron-depleted conditions; lane 6, RT-PCR carried out without reverse transcriptase. (B) Absolute quantification of iupA mRNA and 16S rRNA using RNA isolated from R. equi grown under iron-replete (black bar) or -depleted (white bar) conditions. Insert: relationship between the cycle threshold and number of template molecules used to calibrate the real-time PCR. Target copy number is shown as log₁₀. Shown are the averages of results from three independent experiments in which each sample was analyzed in duplicate.