Computational protocol: Comparative gene expression in toxic versus non-toxic strains of the marine dinoflagellate Alexandrium minutum

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[…] Gene expression was compared between toxigenic (AL3T and AL9T) and naturally non-toxic (AL1T) A. minutum clonal strains originating from the same geographical population in the Gulf of Trieste, Italy (isolated by A. Beran; see []). Bacteria-reduced triplicate cultures were grown under the control conditions as stated above. Culture growth was monitored by daily manual cell counts of samples fixed in Lugol's iodine solution.The toxin content of duplicate samples containing at least 2 × 106 AL3T and AL9T cells and non-duplicate samples containing at least 4 × 106 AL1T cells were analyzed by LC-FD. To confirm the toxin identification of specific PSP toxin analogues by LC-MS, samples of additional parallel cultures were combined into pellets containing 8.5 × 106 to 1.1 × 107 cells.Samples for RNA extraction were taken during exponential growth phase, at 10-11 h (Sampling Time 1, ST1) and at 6-7 h (Sampling Time 2, ST2) after onset of light phase. Total RNA (500 ng sample-1) was extracted as described herein, then amplified and labelled with a Low RNA Input Linear Amplification kit (Agilent, Waldbronn, Germany). The Agilent Low RNA Input Linear Amplification Kit protocol was followed for synthesis of Cy3- and Cy5-labelled cRNA and microarray hybridisation. Microarrays were scanned on an Agilent G2565AA scanner, and raw data were extracted with the Agilent Feature Extraction Software version (FE). Array quality was monitored with the Agilent QC Tool (v1.0) with the metric set GE2_QCMT_Feb07.Pre-processed data were subjected to SAM (Significance Analysis of Microarrays []) as implemented in MeV 4.0 [], and SAM-based q-values [] were calculated. Probes with a q-value of <1% were considered to indicate differential expression of the corresponding genes if the mean fold change of the sample triplicate was at least 1.5. To minimise the influence of physiological differences between the strains that were not related to the capacity for toxin production, only genes identified as significantly higher or significantly less expressed in both toxic strains at both sampling time-points were designated as "higher expressed" or "less expressed" in toxic strains.Samples were clustered by Hierarchical Cluster Analysis (HCL) support trees as implemented in MeV 4.2. Trees calculated with different distance measures (Euclidean, Manhattan and Covariance values) and different distance metrics (average, complete and single linkage) were compared. Node confidence was tested by 1000 Bootstrap replicates.To test reliability of the microarray, expression levels of six genes were evaluated by qPCR. Four of these genes had been identified as significantly higher expressed in the toxic strains, the other two as significantly higher expressed in the non-toxic strain. The qPCR was carried out as described [] with the lepidopteran genes MA and NSP as an artificial internal reference. Primers for qPCR were designed with PrimerExpress 3.0 (Applied Biosystems, Darmstadt, Germany) and synthesized by Eurofins MWG Operon (Ebersberg, Germany). Standard curves using cDNA plasmids corresponding to target sequence ESTs were plotted to test the primer pairs for consistent efficiency at different concentrations. These plasmids were amplified by M13-primed PCR; qPCR primers were tested on dilution series of plasmid PCR products spanning at least 8 orders of magnitude. The qPCR reaction was based on the PowerSybrGreen PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions, using a 7000 Real-Time PCR System (Applied Biosystems).Prior to cDNA synthesis the samples were spiked with the artificial internal control RNAs MA (1 ng reaction-1) and NSP (1 pg reaction-1). Equal amounts of RNA from all strains were processed in parallel. All qPCR reactions based upon the same primer-set were run on the same plate, and reaction efficiencies were compared with MA and NSP-specific primers. To test for contamination with genomic DNA, negative controls consisted of full reactions in which cDNA was exchanged for RNA aliquots of all samples. Thresholds were determined manually for each primer set. Relative expression levels were recorded as the cycle threshold value (Ct). […]

Pipeline specifications

Software tools Agilent Feature Extraction, SAM
Application Gene expression microarray analysis
Diseases Shellfish Poisoning