After the filtering, trimming, and clustering processes the 1,533 obtained ESTs were evaluated based on functional annotation. The cDNA
fragments used to spot the macroarray membrane were amplified by PCR using M13 primers [forward 5'-CAGGAAACAGCTATGAC-3' and reverse 5'-GTAAAACGACGGCCAG-3'] that annealed to selleck kinase inhibitor the vector pDNR-LIB (Clontech), transferred in duplicate to membranes (Hybond N+, Amersham Biosciences) [72] and fixed using a UV crosslinker (Spectronics Corporation). For macroarray hybridization, two distinct RNA pools were used: one cDNA mixture of three distinct biological samples from the initial cultivation phases on artificial media (white phase), and another cDNA mixture of three distinct biological samples from the primordial stage. The membrane was hybridized twice with each cDNA pool. Labeling (400 ng of each cDNA pool), pre-hybridization (4 h), hybridization (2.5 h) and signal detection were performed as recommended by the Eltanexor mouse manufacturer of the Alkaphos kit (GE Healthcare). The membranes were exposed to X-Omat (Kodak) Bafilomycin A1 concentration film for 2.5 h and the images captured using the Scanner Power Look 1120 UDS (Amersham Biosciences) and analyzed with BZ Scan [73]. The presence or absence of the signal, as well as the intensity, was registered for each individual spot. Global normalization and clustering of the generated intensities, using software Cluster version 3.0 [74]. The default Cluster for normalization was performed
eight times, with genes centralized by average. A total clustering of genes was made by the uncentered
method (Pearson correlation). This value used in hierarquical clustering represents the average intensity of each gene. Student’s t-test, was used after global standardization and before clustering to establish a comparison between means. The values significant at 5% probability and the genes accession numbers are shown in Table S1 [see Additional file 1] together with the fold change values based on the means generated triclocarban after normalization by Cluster 3.0 software. Quantitative analyses of reversed transcripts (RT-qPCR) During the growth period in artificial medium, 12 selected genes were analyzed based on their expression pattern derived from the macroarray. The following genes were selected from the EST data base http://www.lge.ibi.unicamp.br/vassoura encoding the proteins: three putative hemolysins (CP03-EB-001-020-G09-UE.F; CP03-EB-001-008-C10-UE.F; CP03-EB-001-024-G03-UE.F), a putative 60S ribosomal L18 protein (CP03-EB-001-001-E05-UE.F), a putative Rho1/GEF (CP03-EB-001-012-F03-UE.F), a putative Rab (Ras family) (CP03-EB-001-020-F11-UE.F), a putative multi-protein-bridging factor (CP03-EB-001-025-E06-UE.F), a putative Ras-GTP-binding protein Rhb1 (CP03-EB-001-005-E11-UE.F), a putative glucose transporter (CP03-EB-001-015-G10-UE.F), a putative cytochrome P450 (CP03-EB-001-025-D09-UE.F), a putative adenylate cyclase (CP03-EB-001-025-C05-UE.