Computational protocol: The Gastric Ganglion of Octopus vulgaris: Preliminary Characterization of Gene- and Putative Neurochemical-Complexity, and the Effect of Aggregata octopiana Digestive Tract Infection on Gene Expression

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[…] Primers were designed by Primer3Plus software (www.bioinformatics.nl/primer3plus) using target sequences deduced from O. vulgaris transcriptome (Petrosino, ). Primer parameters were set to 20 nucleotides in length, product size 100–200 base pairs and melting point 58–60°C. Primers were also analyzed with the Multiple Primer Analyzer to estimate presence of, and possibly avoid, primer-dimers.The efficiency of each pair of primers (Table ) was calculated according to standard methods curves, and following Sirakov et al. (). Briefly, five serial dilutions (1:5, 1:10, 1:20, 1:40, 1:80) of a standard sample were made to determine the efficiency of reactions conducted with each pair of primers. Standard curves were generated for each sample/gene combination using the Ct value vs. the logarithm of each dilution factor (Pfaffl et al., ; Radonić et al., ).Each amplification reaction was conducted in a volume of 25 μl containing: 2 μl of diluted cDNA template, 2.5 μl of 10 × PCR reaction buffer (Roche), 2.5 μl of dNTP mix (0.2 mM), 1 μl of each primer (25 ρmol/μl), 0.25 μl of Taq DNA polymerase (5U/μl), and sterile H2O. The amplification cycles were conducted by Peltier Thermal Cycler PTC-200 (MJ Research). After denaturation at 95°C (2 min) 34 amplification cycles were carried out as follows: denaturation (94°C, 15 s), annealing (60°C, 30 s), extension (72°C, 1 min). Finally, an extension cycle was carried out at 72°C for 7 min to complete all the strands. PCR products were run on 2% agarose gel in TBE buffer 0.5 × (45 mM Tris-borate, 1mM EDTA) and detected expected bands were isolated. DNA was extracted using GenElute Gel Extraction Kit (Sigma-Aldrich, NA1111) and analyzed using an Automated Capillary Electrophoresis Sequencer 3730 DNA Analyzer (Applied Biosystems). [...] In order to analyze expression levels of specific genes of interest, a panel of putative reference genes was first screened to find the most stable genes for these experimental conditions (see the approach utilized for octopus in Sirakov et al., ). In our experiments, we utilized: eukaryotic translation initiation factor 4 (EIF4G1), LIM and SH3 domain protein (F42H10.3), lamin-B1 (Lmnb1), cytoplasmic FMR1 (Sra-1), ubiquitin-40S ribosomal protein S27a (RPS27A), elongation factor 1-alpha (eef1a), 40S ribosomal protein S18 (RPS18). The gene expression stability of the candidate reference genes for our samples was evaluated with BestKeeper (Pfaffl et al., ) and NormFinder (Andersen et al., ), following Sirakov et al. (). We identified the three most stable reference genes as Lmnb1, Sra-1 and RPS27A.For gene expression experiments, samples from 10 octopuses were processed in triplicate. Polymerase chain reactions were carried out in an optical 384-wells plate with Applied Biosystems ViiA7 (Life Technologies) using Fast SYBR Green Master mix (ThermoFisher Scientific) to monitor dsDNA synthesis. Reactions (total volume: 10 μl) contained: 1 μl cDNA, 5 μl SYBR Green Master mix reagent, 4 μl of forward and reverse primers mix (0.7 pmol/μl each). The following thermal profile was used: 95°C for 10 min; 95°C for 15 s, 60°C for 1 min, 40 cycles for amplification; 72°C for 5 min; one cycle for melting curve analysis, from 60° to 95°C to verify the presence of a single product. Specificity of PCR products was checked by melting curve analysis followed by gel electrophoresis and DNA sequencing. PCR data were analyzed using the ViiA™ 7 Software (Life Technologies) to determine cycle threshold (Ct) values. Each assay included a no-template control for every primer pair. All sequences have been deposited in GenBank after validation (Table ). […]

Pipeline specifications

Software tools Primer3, NormFinder
Application qPCR
Organisms Octopus vulgaris, Toxoplasma gondii
Diseases Infection, Stomach Neoplasms
Chemicals Acetylcholine, Dopamine, Felypressin, gamma-Aminobutyric Acid, Glutathione, Norepinephrine, Octopamine, Serotonin, Superoxides, Tachykinins