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Sample GSM6675244

Query DataSets for GSM6675244

Status

Public on Nov 01, 2023

Title

Gill, Survivor, Day 0 (Fp_40_G)

Sample type

RNA

Source name

Survivor

Organism

Oncorhynchus mykiss

Characteristics

replicate: Replica 6
tissue: Gill
infection: Flavobacterium psychrophilum
time: Day 40
outcome: Survivor

Treatment protocol

Tissue (liver, spleen, gills) samples were fixed in RNAlater (cat.no R0901, Sigma-Aldrich, Denmark), placed at 4°C for 24 h and subsequently stored at -20°C until further processing.

Growth protocol

The fish were kept in separate aerated INTEX® plastic tanks in two different rooms (one room for infected fish, one room for non-infected controls). The tank dimensions were (2.6 m x 1.6 m x 0.65 m, total volume 2.7 m3) and each tank contained 800 L of municipal water. Internal biofilters (20 L min-1 EHEIM, Germany) were placed in both tanks to maintain water quality (pH 7.6, nitrite <0.01 mg/L, nitrate <50 mg/L (TetraTest 6in1, cat.no. T704154, Eldorado A/S, Denmark), ammonia <0.5 mg/l (API Ammonia Test Strips, VioVet Ltd., United Kingdom)). Temperature was kept at 11-12°C, and the light/dark cycle was 12h light /12h dark. The fish received commercial pelleted dry feed (1% biomass daily) (INICIO 917, BioMar A/S, Brande, Denmark).

Extracted molecule

total RNA

Extraction protocol

RNAlater fixed samples from fish (gills, liver and spleen) were homogenized (Tissue-lyser II, Qiagen, Denmark) using a homogenization buffer with 2-mercaptoethanol (Sigma-Aldrich). RNA was extracted using the GenEluteTM mammalian RNA kit (RTN350, Sigma-Aldrich, Denmark). Liver samples were pre-treated by Proteinase K (cat.no.P4850, Sigma-Aldrich, Denmark). DNase I (AMPD1, Sigma-Aldrich, Denmark) treatment was applied to remove genomic DNA. The concentration of RNA was determined in a NanoDrop 2000 spectrophotometer (Saveen & Werner, Denmark). Quality of RNA was evaluated by agarose (cat.no. 16500100, ThermoFisher Scientific, Denmark) gel electrophoresis. RNA was kept at −80°C until cDNA synthesis.

Label

FAM

Label protocol

cDNA synthesis was performed in T100 thermocycler, Biorad, Denmark, using Oligo d(T)16 primer and TaqMan® Reverse Transcription Reagents (cat.no. N8080234, Thermo Fischer Scientific, Denmark). Finally, cDNA was stored at −20°C until further use. Quantitative PCR assays were performed using an AriaMx Real-Time PCR machine (cat.no.G8830A-04R-010, AH diagnostics AS, Denmark). The cycling conditions were one cycle of predenaturation at 95°C for 3 min. This was followed by 40 cycles of denaturation at 94°C for 5 sec with a combined annealing/elongation process at 60°C for 15 sec with endpoint measurement. Primers and Taq-Man probes targeting immune-relevant rainbow trout genes (total of 28 genes including three housekeeping genes) were synthesized at TAG Copenhagen AS, Denmark. Reaction volumes were 12.5 μl (2.5 μl cDNA, 6.25 μl Brilliant III Ultra-Fast QPCR Master Mix (600881, AH Diagnostics AS, Denmark), 1.0 μl primer-probe mixture (forward primer, 10 μM and reverse primer, 10 μM), Taq-Man probe (5 μM), and 2.75 μl RNase-free water. Reverse transcriptase minus and negative controls were used for every plate setup. . NormFinder (Andersen et al., 2004) was applied to evaluate all combinations of three reference genes (Elongation factor (ELF) 1-α, β-actin and acidic ribosomal phosphoprotein P0 (ARP)) as endogenous control; the average of the three genes was chosen and stability values were 0.002, 0.005 and 0.002 for liver, spleen and gills, respectively. In order to quantify the bacterial load in different organs at the different time points we measured the gene expression level of the bacterial gene recA, which was described as a household gene in V.anguillarum (GenBank ass.no. LC370212) by Crisafi et al. (2014). We designed the probe for the qPCR assay according to Zuo et al. (2020) with minor modifications. For efficiency determination, estimated 102%, dilution series of samples with high expression of V. anguillarum recA was used. Specificity was tested by SYBER green and melting curve analysis confirmed by electrophoresis, product length 248 bp. Bacterial transcript level was estimated as 2-ΔCq . It should be noted that dead or inactive bacteria may not be detected by this method.

Hybridization protocol

n/a

Scan protocol

n/a

Description

Fp_40_G

Data processing

All q-PCR assays exhibited efficiencies within 100% ± 5% so the simplified 2-ΔΔCq method was used for analysing data (Livak and Schmittgen, 2001). For normalization and as internal calibrator the average of three genes (ARP, ELF1α and β-actin) was chosen by using NormFinder (Andersen et al., 2004). For gene expression analyses, all challenged groups were compared to non-exposed time point controls using Student’s t-test. We considered differences to be significant when regulations were at least two-fold and p < 0.05. For four of the genes investigated in the non-exposed timepoint control groups less than three fish showed Cq values (excluding a t-test) and therefore we applied a qualitative assessment (presence/absence of Cq values) and analyzed data with the nonparametric Mann-Whitney test using a probability level of 5%.
Target gene signals were normalized using the average of 3 housekeeping genes (ARP, b-actin and ELF 1a) as2^-ΔCt, where -ΔCt = -(Ct_Target − Ct_ average of 3 HKGs). Folds were calculated as 2^-ΔΔCt, where -ΔΔCt = -[ΔCtinfected -ΔCtUninfected] using idividual uninfected time point controls. No fold changes was calculated for samples timepoint zero since there were no infected group at that timepoint.

Submission date

Oct 25, 2022

Last update date

Nov 01, 2023

Contact name

Per Walter Kania

E-mail(s)

pwk@sund.ku.dk

Organization name

University of Copenhagen

Department

Department of Veterinary and Animal Sciences