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Abstract:
The dataset shows quantity and quality of isolated RNA and transcript levels of selected genes in killer whale fibroblasts exposed to pollutants. A fibroblast culture was established from a killer whale skin/blubber biopsy collected in November 2020 from Lyngen, Norway. The cells of passage 3-4 were exposed to a mixture of pollutants over 48 hours. The applied pollutant concentrations were 0.1X, 1X and 10X (column measurementID) i.e., 1.16 µM, 11.57 µM and 115.7 µM, respectively, where 1X reflected median levels of the ten most abundant persistent organic pollutants in killer whale blubber from Northern Norway (p,p’-DDE > PCB-153 > PCB-138 > trans-nonachlor > PCB-180 > PCB-101 = PCB-52 > PCB99 > PCB187 > PCB-118). The selected genes include aryl hydrocarbon receptor (AHR), cytochrome P450 1A (CYP1A), cytochrome P450 3A (CYP3A), cytochrome P450 4A (CYP4A), thyroid hormone receptor alpha (THRA), thyroid hormone receptor beta (THRB), glucocorticoid receptor (GR), peroxisome proliferator activated receptor alpha (PPARA), peroxisome proliferator activated receptor gamma (PPARG), fatty acid binding protein 4 (FABP4), estrogen receptor alpha (ERA), adiponectin (ADIPOQ), cluster of differentiation 36 (CD36) and the two housekeeping genes (HKG) were glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and tyrosine 3-monooxygenase (YWHAZ).
Quality
RNA was isolated from the cells exposed to POP mixtures or solvent control (0.05% DMSO). The RNA yield (ng/µL) and purity (the absorbance ratios at 260 nm and 280 nm (A260/280), and 230 nm (A260/230))were determined using Thermo Scientific NanoDrop 2000 Spectrophotometer. Assessment of RNA integrity number was conducted using RNA 6000 Nano Kit and the assay class Eukaryote Total RNA Nano for the Agilent 2100 Bioanalyzer. Transcript levels of 13 genes of interest were analyzed in the exposed killer whale cells using digital droplet PCR (ddPCR). The ddPCR was performed following the protocol for the QX200™ ddPCR™ EvaGreen Supermix (Bio-Rad). Each reaction was prepared in a total volume of 20 µL: 10 µL of EvaGreen Supermix (2X), 0.2 µL of each primer (10 µM), 5.1 µL of nuclease-free H2O, and 4.5 µL of cDNA (1.875 ng/µL; synthetized from the isolated RNA). The transcript levels of genes are given as the absolute value of cDNA concentration expressed in copies/µl for each sample.