. When RT-PCR was used to assess the reliability of the microarray hybridizations germlings were exposed to a novel growth curve (new RNA samples, not stocks of the original RNA used in the array experiment). Real-time RT PCR reactions All the PCR and RT-PCR reactions were performed using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, USA). Taq-Man™ Universal PCR Master Mix kit (Applied Biosystems, USA) was used for PCR reactions. The reactions and calculations were performed according to Semighini et al. . The primers and Lux™ fluorescent probes (Invitrogen, USA) used in this work are described in Additional file LY294002 research buy 4, Table S3. Staining and microscopy For cell imaging of RcnA fused to GFP, conidiospores
were grown in glass-bottom dishes (Mattek Corporation, USA) in 2 SB202190 mouse ml of MM+2% glycerol for 24 hours at 30°C. All the confocal images were analysed using the Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems, Heidelberg, Germany) (Laboratory of Confocal Microscopy, FMRP-USP, Brazil) using 63× magnification water immersion objective lens using laser lines 488 nm for GFP and 405 nm for DAPI. Images were captured by direct acquisition with the Leica LAS
AF software (Leica Microsystems) and additional processing was carried out using Adobe Photoshop 7.0 (Adobe Systems Incorporated, CA). DNA manipulations and construction of the Aspergilli conditional mutants DNA manipulations were according to Sambrook and Russell . All PCR reactions were performed using Platinum Taq DNA Polimerase High Fidelity (Invitrogen). For the DNA-mediated transformation, the deletion cassettes were constructed by “”in vivo”" recombination in S. cerevisiae as previously described by Colot et al. . About 2.0-kb regions on either side of the ORFs were selected for primer design. For the construction of the A. fumigatus rcnA deletion, mafosfamide the primers calp-Afu P1 and calp-Afu P2 were used to amplify the 5′-UTR flanking region of the targeted ORF. The primers calp-Afu P3 and calp-Afu P4 were used to amplify the 3′-UTR ORF flanking region. For
the construction of the A. nidulans rcnA deletion, the primers calp-Ani P1 and calp-Ani P2 were used to amplify the 5′-UTR flanking region of the targeted ORF. The primers calp-Ani P3 and calp-Ani P4 were used to amplify the 3′-UTR ORF flanking region. Both fragments 5- and 3-UTR were PCR-amplified from genomic DNA using as templates the A4 CHIR98014 strain for A. nidulans and AFU293 for A. fumigatus cassettes. The pyrG used in the Aspergilli cassettes for generating both deletion strains was used as marker for auxotrophy and were amplified (by using primers pyrG Fw and pyrG Rw) from pCDA21 plasmid . Cassettes generation was achieved by transforming each fragment for each construction along with the plasmid pRS426 BamHI/EcoRI cut in the in S. cerevisiae strain SC9421 by the lithium acetate method . The DNA of the yeast transformants was extracted by the method described by Goldman et al.