island of origin: Ålgrundet family: 6 tissue: liver treatment: decreasing water developmental stage: Gosner Stage 37
Treatment protocol
Tadpoles originating from each island were raised to Gosner stage 23 (active swimming) (Gosner, 1960) and then one set was reared in constant water while a second set was reared under reduced water conditions. Four tadpoles from each female (two allocated to each treatment) were randomly chosen and placed into individual plastic containers (9.5 cm × 9.5 cm, height 10 cm). The containers were filled with 750 mL of tap water, previously aged and aerated together with dried deciduous leaves. The leaves were removed when the water was transferred to the experimental containers. The tadpoles were subjected to one of two treatments, either constant water level (C) or simulated pool drying (D). In the simulated drying treatment the initial water volume of 750 mL was lowered by 33% every fourth day, starting at day 5 and continued until day 25, after which the water volume in the D treatment was kept constant at 66 mL. The water temperature did not differ between the two treatments. The experiment was terminated at Gosner stage 37 (front legs visible), after which tadpoles were weighed. Liver samples were obtained from individuals in all groups at a fixed time point 14 days post commencement of water adjustment and when animals had reached Gosner stage 37.
Growth protocol
Rana temporaria eggs were collected from six islands within the Gulf of Bothnia, northern Sweden. For each island, samples of 20–50 eggs were taken from a maximum of 10 clutches and transported to the laboratory. Each clutch was assumed to represent one female, as female R. temporaria lay only one egg clump per season. Eggs from each female were kept in separate containers filled with aged tap water. The eggs were stored in thermo-constant laboratory conditions and kept cool (4 °C) until all eggs were collected. When all eggs were collected, the temperature of the laboratory was set to 22 °C for the remainder of the experiment. A light : dark cycle of 18 h : 6 h was employed. The tadpoles were fed ad libitum every fourth day on a mixture (1 : 2) of finely ground fish food and rabbit chow. The food mass was 15 mg per tadpole at the beginning of the experiment, increased to 30 mg at day 9, 45 mg at day 13, 60 mg at day 17, and 75 mg from day 21 and to the end of the experiment. Water was replaced every fourth day, prior to feeding.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from RNAlater (Life Technologies) preserved liver tissue using TRIzol (Life Technologies) and a Retsch MM301 mixer mill with a 3mm tungsten carbide bead and 1 ug used to prepare amplified cDNA using the SMARTer cDNA synthesis kit (Clontech Laboratories). SMARTer cDNA samples were purified using the GeneJET PCR Purification Kit (Fermentas Canada).
Label
Cy3
Label protocol
Amplified cDNA (500 ng) was random labeled with 2 ul of 1 mM Cy3-dCTP (GE Healthcare) using the DecaLabel DNA Labeling Kit (Fermentas Canada).
Hybridization protocol
Prepare a MAGEX slide by hydration in WB2 buffer (1xSSC, 0.1% SDS) at ambient temperature followed by incubation in PB1 buffer (5xSSC, 0.2% BSA, 0.2% SDS) for 45 minutes at 48°C. Rinse the slide with water followed by immersion in isopropanol at ambient temperature and centrifuge dry. Place a Lifterslip CS/3 (Erie Scientific Company) over each array region on the slide. Combine the Cy labeled sample (5.5ul of 15 pmol/ul Cy3 label) and reference (1 ul of 2 pmol/ul Cy5 label) cDNA and briefly denature at 95°C for 3 min followed by immediate incubation on ice. Prepare a 33 ul hybridization mix containing 5xSSC, 25% deionized formamide, 0.02% BSA, 1ug A45 oligonucleotide, 0.1% SDS, 5% Dextran, and 6.5 ul of the combined Cy3-labeled sample and Cy5-labeled reference cDNA. Briefly warm the hybridization solution at 37°C and then place onto the MAGEX array and incubate the prepared slide in a Hybex incubator at 42°C for 18 hrs. Hybridized slides were washed once with WB1 buffer (1xSSC, 0.1% SDS) at 48°C for 5 min; twice with WB2 buffer (0.1x SSC, 0.1% SDS) at 48°C for 10 min; and once with WB3 (0.01x SSC, 0.1% SDS) at 48°C for 10 min. Dry the slides by centrifugation at 200 x g and store in a desiccator prior to data collection.
Scan protocol
Scanning was done on a Packard Bioscience Scan Array Express Micro array scanner with the Cy3 PMT gain at 80% and Cy5 PMT gain at 70%. Laser strength was set at 90% for both channels. MAGEX 2008 microarray slides were scanned at 5 um resolution with an X=0 mm, Y=11.73 mm, W=22 mm, and H=52.38mm.
Data processing
Data was aquired from raw scanned MAGEX 2008 array images using ImaGene v9.0.
Evaluation of phenotypic plasticity in Rana temporaria tadpole development
Data table header descriptions
ID_REF
VALUE
Imagene V9.0 computed signal were normalized using correction factors derived for each array from spot signals greater than 500 and representing genes displaying no statistically significant differences between treatment groups as determined by Kruskal-Wallis analysis.
