Nt tissue sample, 2 g of stem PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26240184 segments (four plants) were Imatinib

Nt tissue sample, 2 g of stem PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26240184 segments (four plants) were Imatinib (Mesylate) web excised with a sterile razor blade, dehydrated in liquid nitrogen and stored at -80 . Total RNA was extracted using TRIzol reagent (Invitrogen) and treated with DNase (Sigma-Aldrich) following the manufacturer’s instructions. For fungal colonies, total RNA was extracted from frozen single-spore colonies (60-150 mg), grown for 8 days on PDA at 24 , with the RNeasy Mini kit (Qiagen) following the manufacturer’s protocol for plant tissues.cDNA-AFLP analysisWe used the cDNA-AFLP protocol described by Vos et al. [82] and Bachem et al. [83] with the modifications and primers described by Breyne et al. [84] which permit the visualization of a single cDNA fragment for each mRNA present in the original sample, reducing the output sequence redundancy. Double-stranded cDNA was synthesized from 2.5 g total RNA using the Superscript II reverse transcription kit (Invitrogen) and a biotinylated oligo-dT primer (Promega). After pre-amplification, the mixture was diluted 600fold and 5 l was used for selective amplification with 128 primer combinations, carried out with one selective nucleotide added on the 33P-labeled BstYI primer and two selective nucleotides on the MseI primer. We used the following touch-down PCR conditions: 2 min denaturation at 94 followed by 30 s denaturation at 94 , 30 s annealing at 65 , 60 s extension at 72 for 13 cycles (scaledown of 0.7 per cycle); 30 s denaturation at 94 , 30 s annealing at 56 , 60 s extension at 72Sestili et al. BMC Genomics 2011, 12:122 http://www.biomedcentral.com/1471-2164/12/Page 18 offor 23 cycles, and 5 min at 72 . Selective [g-33P]ATPlabeled amplification products were separated on a 6 polyacrylamide gel in a Sequi-Gen GT Sequencing Cell (38 ?50 cm) (Bio-Rad) running for 2.5 h at 115 W and 50 . Gels were dried onto 3 MM Whatman paper for 2 h at 80 on a Gel Dryer Model 583 (Bio-Rad) and marked with Glogos II Autorad Markers (Stratagene) before exposing to Kodak Biomax MR films, for 12-16 h.Sequence analysis of cDNA-AFLP fragmentsBands corresponding to differentially expressed genes were excised from the gels with a surgical blade and the eluted DNA was reamplified using non-labeled primers identical to those employed for selective amplification and the following PCR conditions: 15 min denaturation at 94 , 40 s denaturation at 94 , 60 s annealing at 56?C, 40 s extension at 72 for 35 cycles, and 5 min at 72?C. The quantity of each reamplified bands were checked on a 1.8 agarose gel against the 1650-bp fragment of the DNA ladder 1 Kb plus (Invitrogen). PCR products were purified with MultiScreen PCR 96 plates (Millipore) and sequenced directly (BMR Genomics). Sequence information was obtained by comparing nucleotide and protein sequences in the available public databases by BLAST sequence alignment [85]. Homology searching was carried out against the following databases: NCBI [34], Cucurbit Genomic Database Melon Unigene ver. 4.0 [32], UNIPROT database [33] and Fusarium Comparative Database [39]. Sequences were manually assigned to functional categories based on the analysis of scientific literature and also with the aid of the information reported for each sequences by the Gene Ontology Consortium [35], where available. Sequence data from this article have been deposited in GenBank, Accession Numbers: HO867279- HO867981.Real-time RT-PCR analysiswith ROX (Invitrogen). All samples were examined in three technical replicates. Experiments wer.