Groups of twenty juveniles < 24 h old were exposed to selected exposure levels in 0.5 L of ASTM hard water with food present (5x105 cells/mL of C. vulgaris) during 3 consecutive days. Contaminants were dosed using acetone as a carrier (0.1 ml/L). Treatments were quadruplicated. At the end of exposure the 20 juveniles of each treatment per replicate were collected, pooled in an eppendorf, flash- frozen in liquid N2 and preserved at -80 oC until RNA extraction.For adult reproductive tests, exposures started with 8-9 day old gravid females, which were exposed during three consecutive broods to the studied chemicals (10-14 days). Just after releasing their fourth clutch into the brood pouch, eggs were gently flushed from the brood pouch and females were immediately flash- frozen in liquid N2 and preserved at 80oC until RNA extraction.
Growth protocol
All experiments were performed using a well-characterized single clone of Daphnia magna (Clone F), maintained indefinitely as pure parthenogenetic cultures. Individual or bulk cultures of 10 animals/L were maintained in ASTM hard synthetic water. Individual or bulk cultures were fed daily with Chorella vulgaris Beijerinck (5x105 cells/mL, respectively, corresponding to 1.8 mg C/mL; [24]). C. vulgaris was grown axenically in Jaworski/Euglena gracilis 1: 1 medium (CCAP, 1989). The culture medium was changed every day, and neonates were removed within 24 h. Photoperiod was set to 14h light: 10h dark cycle and temperature at 20 ± 1oC.
Extracted molecule
total RNA
Extraction protocol
Qiagen Rneasy
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.4 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
600 ng of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the custom-designed Agilent test array for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent), 1 minute with 37°C GE Wash buffer 2 (Agilent), 30 seconds with Acetonitrile, and dried with Agilent Stabilization and Drying Solution.
Scan protocol
Slides were scanned immediately after washing on a GenePix 4200AL scanner at PMT gain level 420 using default setting (Scan resolution 5um, One channel-Green, Laser power 100%).
Data processing
The scanned images were analyzed with GenePix Pro 6.1 (Molecular Devices, Sunnyvale, CA) using default parameters and Agilient-provided GEML grid file to obtain signal intensities. GeneSpring GX (Agilent Technologies) was used to normalize data by Quantile normalization and baseline transformation to the median of all samples. Statistical analysis was also performed in GeneSpring utilizing data from all microarray probes and consisted of one-way ANOVA with Benjamini-Hochberg multiple-tests correction to test for significant differential expression of microarray targets.
Identification of metabolic pathways in Daphnia magna exposed to selective serotonin reuptake inhibitors using transcriptomic, immunocytochemistry and physiological responses.
