Clc genomics workbench 12 software#
Threshold line was set manually at 0.2 in every analysis, performed using the Sequence Detection Software v. Melting curve analysis was performed to confirm the specificity of the amplification product.
Clc genomics workbench 12 plus#
Reaction mix (10 m L reaction) comprised 1 6 Hot FirePol EvaGreen qRT-PCR Mix Plus (ROX) buffer (Solis BioDyne, Tartu, Estonia), 10 m M forward and reverse primer pairs and 3 m l (201.3 6 39.3 ng m l -1 ) cDNA. Amplification conditions were 15 min at 95 u C, followed by 45 cycles of 30 s at 95 u C and 1 min at 60 u C.
Real-time quantitative PCR analyses were then performed on the ABI 7500 Fast Real-Time Sequences Detection System (Applied Biosystems, Foster City, CA, USA). The PCR products were then loaded on a 1% (w/v) agarose gel in 0.5 Tris-Borate-EDTA (TBE) buffer at 125 Volts for 1.5 h. PCR thermo-cycler conditions were: 3 min at 94 u C, followed by 35 cycles of 94 u C for 30 s, 60 u C for 30 s, and 72 u C for 30 s. The Mastermix comprised 1 6 PCR Buffer (Fermentas, Hilden, Germany), 0.1 mM dNTPs, 0.05 m M primer pairs, 0.07 U m l -1 DreamTaq DNA Polymerase (Fermentas) and 5 m l (20.1 6 3.9 ng m l -1 ) cDNA. Preliminary specificity tests were performed using the end-point PCR performed with each of the five primer pairs (MT3, LOX, LTP, PX and EDS, File S1) in a 20 m l reaction. The concentration of cDNA samples was quantified using a NanoDrop ND-8000 spectrophotometer (Thermo Scientific, Wilmington, USA). x domestica ‘Golden Delicious’ uninoculated L1 and L7 leaves collected at 72 and 96 hpi) were reverse-transcribed in triplicate using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Fermentas, Hilden, Germany), following the manufacturer’s instructions.
1.0 using the BLAST function of the Genome Database for Rosaceae (GDR: Independent RNA samples ( M. gov/tools/primer-blast) of the National Center for Biotechnology Information (NCBI: ) and verified against Malus x domestica genome v. Specific primers for the five candidate genes were designed (File S1) using the Primer-BLAST webtool (.
The analysis focused on signalling and hormone pathways, genes encoding chemical defences like pathogenesis-related proteins, genes encoding physical barriers like cuticle, waxes and callose, genes acting on the biosynthesis or transport of substances connected to fungal nutrition and genes regulating the acidity of leaf tissues like proton transporters and cation/anion co- transporters. KEGG pathway maps were then enriched by inserting the most significant DEGs found with the MapMan webtool. Pathway analysis was performed using the KEGG function of the Blast2Go webtool. Default parameters were used and JGI Chlamy Augustus models, TIGR5 rice proteins, InterProScan, and a Blast cut-off of 50 were selected. Moreover, a fasta file with all DEGs was generated and sent through the Mercator webtool ( guest/app/mercator) for Bincode mapping, and through webtool MapMan v.3.5.1 ( mapman, ) for pathway analysis. Sequences were then loaded on Blast2Go v.2.5.1 for gene ontology functional annotation using level 2 GO vocabulary for biological process terms, molecular function terms, and cellular component terms. The 20 most abundant transcripts for each cDNA library were filtered using the RPKM normalisation procedure on the CLC Genomic Workbench. 2.5.1 ( ) for Blastx and gene ontology analysis, separated using the Gene Ontology (GO) vocabulary ( Ontology annotations were then refined using InterPro Scan, ANNEX, GoSlim and KEGG (Kyoto Encyclopaedia of Genes and Genomes functions of the Blast2Go platform. The resulting DEGs were then loaded on Blast2Go v. Identification of DEGs was based on normalised gene expression calculated as RPKM, analysed using the Baggerley’s test and filtered with the False Discovery Rate (FDR) P -value correction of 0.0001 (one false discovery in 10000 discoveries). component analysis with all differentially expressed genes (DEGs) found in each cDNA library.
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