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. 2012 Dec 26;7(12):e52734. doi: 10.1371/journal.pone.0052734

Figure 5. Silencing of the reporter gene URA5-miRs.

Figure 5

(A) The upper panels show a slower growth rate of the transformants of URA5-miR1 (two transformants was picked in each case, namely, miR1-1 and miR1-2), and URA5-miR2 (miR2-1 and miR2-2), than the wild type B4500, and the transformants of miR1-m (i.e. miR1-m1 and m2) and miR2-m, on MIN agar supplemented with 100 µg/ml hygromycin B, no hygromycin for B4500 and B4500FOA. The negative control B4500FOA (ura5) did not grow on MIN. On MINFOA (the bottom panels), the wild type strains and transformants of miR1-m and miR2-m were killed by FOA. Transformants containing miR1 and miR2, as well as ura5 − strain B4500FOA grew properly. MIN: minimal medium, and MINFOA: minimal medium with 5-FOA and 50 mg/l uracil. For selection of transformants, 100 µg/ml hygromycin B was added to MIN or MINFOA when needed. (B) Reverse transcription-PCR confirmed the decreased mRNA level of URA5 in URA5-miR transformants. URA5 mRNA levels in the wild type and in the transformants of miR-ms were close to each other. In each assay, two transformants were picked for double examination. (C) A semi-quantification of URA5 mRNA to ACT1 mRNA in the strains in (B).