tissue: brain treatment: nAg (6 µg/L nominal Ag; Vive Nano, Toronto, ON) with vehicle
Treatment protocol
The exposure setup was modified from the Organization for Economic Co-operation and Development (OECD) Amphibian Metamorphosis Assay (AMA) guidelines (2009). Twenty four tadpoles were immersed for 6 d in WW, nAg (6 µg/L nominal Ag; Vive Nano, Toronto, ON), or iAg (6 µg/L nominal Ag; AgNO3, Fisher Scientific). On experimental day 4, half the tadpoles in each immersion condition (n=12) received a 0.1% (0.01 ml/g body weight) intraperitoneal injection of TH (10 nM 3,3′,5-triiodo-L-thyronine; Sigma- Aldrich) prepared in 100 nM NaOH, while the remaining half were injected with vehicle alone. At 48 h post-injection (experimental day 6), animals were euthanized in 0.1% (w/v) tricaine methanesulfonate (MS-222; Syndel Laboratories Ltd, Vancouver, BC) buffered with 25 mM NaHCO3 and brain and liver tissues collected and immediately preserved in RNAlater (Qiagen) at 4°C followed by long term storage at -20°C.
Growth protocol
Animals used in the present study were treated and maintained in accordance with the guidelines of the Canadian Council on Animal Care and the Animal Care Committee guidelines of the University of Victoria. The exposures were carried out at the Pacific Environmental Environment Centre (PESC) in Vancouver, BC using locally wild-caught R. catesbeiana tadpoles ranging between Taylor-Kollros developmental stages IV and X. Animals were maintained in 35 L tanks filled with well water (WW) for the duration of the exposures. Water temperature was maintained at 20.41 ± 0.07°C with 84.2 ± 0.4% dissolved oxygen and a pH of 7.78 ± 0.01. The photoperiod was kept at 12 hrs for both light and dark periods. The exposure was a static renewal system and water + treatmentswere replaced every 2-6 d followed by tadpole feeding of algal pellets. Nitrogenous wastes were monitored and kept within non-toxic levels for ammonium (15 ± 5 μg/L), nitrite (13 ± 3 μg/L), and nitrate (71 ± 18μg/L).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from brain and liver tissues using TRIzol reagent (Invitrogen) according to manufacturer’s protocol. Each sample was homogenized at 20 Hz for 6 min in 700 µl TRIzol using a 3 mm tungsten-carbide bead and a Retsch MM301 mixer mill. RNA pellets were resuspended in 100 µl (liver) or 40 µl (brain) diethyl pyrocarbonate-treated water, quantified by spectrophotometry, and stored at -80°C. Each total RNA sample was used to prepare amplified cDNA with the SMARTer PCR cDNA Synthesis Kit as per manufacturer’s protocol (Clontech Laboratories Inc.) with subsequent cDNA purification using the GeneJET PCR Purification Kit (Fermentas). All cDNA was stored at -20°C.
Label
Cy3
Label protocol
The DecaLabel DNA Labelling Kit (Fermentas) was used to incorporate Cy3-dCTP (GE Healthcare Bio-Sciences Corp.) into cDNA according to manufacturer’s specifications. To aid in microarray data collection, a universal DNA reference (UDR) containing amplicons representative of all gene sequences present on the MAGEX microarray was similarly prepared with Cy5-dCTP (GE Healthcare Bio-Sciences Corp.). Labelled DNA was purified with the GeneJET PCR Purification Kit, and sufficient fluorophore incorporation was determined with the Nanodrop UV-Vis spectrophotometer (ND-1000, Thermo Scientific). Labelled DNA was precipitated with sequential addition of 20 μg glycogen (ThermoFisher Scientific), 5 µl of 3 M sodium acetate (pH 5.2), and 150 µl of chilled 100% ethanol. Samples were incubated at -20°C for a minimum of 60 min followed by centrifugation at 13,000 x g at 4°C for 20 min. The DNA pellet was washed once with 300 µl 75% ethanol and air dried for 10 min prior to addition of 7 µl (tadpole tissue samples) or 30 µl (UDR) dH2O, and stored at -20°C in 1.5 ml amber tubes. For hybridization, Cy5-labeled UDR (1 µl) was mixed with each Cy3-labeled cDNA (5.5 µl) and incubated at 95°C for 3 min followed by cooling on ice and a brief centrifugation to collect the denatured material. For each sample, a 33 µl hybridization solution was prepared that contained 5x SSC, 25% (v/v) deionized formamide, 0.02% (w/v) BSA, 1 µg A45 oligonucleotide, 0.1% (w/v) SDS, 5% (w/v) dextran, and 6.5 µl denatured labelled DNA and warmed briefly at 37°C prior to application onto the covered MAGEX DNA array.
Hybridization protocol
MAGEX DNA array slides were briefly rinsed in a plastic coplin jar containing WB2 buffer (0.1x SSC, 0.1% (w/v) SDS) and subsequently incubated with 90 ml of PB1 buffer (5x SSC, 0.2% (w/v) BSA, and 0.2% (w/v) SDS) at 48°C for 45 min. Each slide was dried by centrifugation in a 50 ml falcon tube for 3 min at 200x g. Dried Lifterslip CS/3 coverslips (Erie Scientific Company) prerinsed with dH2O and 100% ethanol were placed over each DNA array region and the preheated cDNA/UDR samples added to each array. MAGEX DNA array slides were placed in a slide rack and 300µl of dH2O was added to the humidity filter prior to chamber assembly. Hybridization was done for 18 h at 42°C in a Hybex Microsample Incubator (SciGene). All buffers used in washing the microarry slides were prewarmed at 48°C prior to use. Following hybridization, coverslips were removed and each slide washed for 5 min with WB1 buffer (1x SSC and 0.1% (w/v) SDS). Two subsequent washes were performed with WB2 buffer followed by a wash with WB3 buffer (0.01x SSC and 0.1% (w/v) SDS). Slides were dried by centrifugation for 3 min at 200x g and stored in the dark in a Scienceware autodessicator.
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. Spot intensity levels were obtained via ImaGene Version 9.0 (BioDiscovery Inc.) as per the manufacturer’s protocol using the UDR to align the spot grid.
Data processing
Data normalization involved use of the geometric means derived from spot values representing high quality gene signals obtained for each sample. This resulted in normalization factors between 1.00 to 1.95 (brain) or 1.00 to 4.36 (liver). An appropriate background signal was determined by evaluating the empty and buffer-only spot positions interspersed throughout each array. The universal floor value was set at 1 robust standard deviation greater than the highest median background signal across all arrays, helping to minimize false positives due to low signal intensities.The floor was 326 and 166 for brain and liver, respectively.
Impact of exposure to environmentally-relevant concentrations of ionic silver and nanosilver on thyroid hormone-dependent postembryonic development of the North American bullfrog (Rana catesbeiana)
Data table header descriptions
ID_REF
VALUE
Imagene Version 9.0 computed, normalized signal intensities with universal floor applied.
