Morphine (morphine hydrochloride; Polfa, Kutno, Poland) was administered subcutaneously (s.c.). Experimental groups (control, acute and chronic morphine) consisted of 9 animals from each strain. To obtain the most reliable comparison, control and acute morphine groups received injections of saline for four days in the same time schedule as the chronic group received morphine. On the fifth day, acute treated animals were injected with a single dose of morphine (20 mg/kg) and killed by decapitation after 4 h. Chronically treated animals were injected with increasing doses of morphine for five days. Mice received morphine thrice daily (09:00 h, 13:00 h and 17:00 h) for four days using a dosing schedule of 10, 20, 40 and 40 mg/kg of morphine on days 1, 2, 3 and 4, respectively. On the last day, a final morphine dose of 40 mg/kg was administered and 4 h after the last injection, the animals were sacrificed. Mice in control groups were killed 4 h after the last injection of saline. The dose scheme and time schedule were used in order to maximize strains differences in response to morphine.
Growth protocol
Adult male (8 to 10 weeks of age) 129P3/J (000690), DBA/2J (000671), C57BL/6J (000664) and SWR/J (000689) mice (The Jackson Laboratory, Bar Harbor, Maine, USA) were housed 6 per cage, under a 12 h dark/light cycle, with ad libitum access to food and water. Animals weighing from 20 to 30 g were used throughout the experiments. The animal protocols used in the study were approved by the local Bioethics Commission working at the Institute of Pharmacology, Polish Academy of Sciences (Krakow, Poland).
Extracted molecule
total RNA
Extraction protocol
Trizol/RNeasy extraction of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Following labeling, samples were hybridized to the GeneChip Test3 array (Affymetrix, Santa Clara, CA) for quality control. Fragmented cRNA (15 μg) was used for hybridization to the GeneChip Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA). Arrays were washed and stained with streptavidin-phycoerythrin (Merck, Darmstadt, Germany) in Fluidic Station 400 (Affymetrix, Santa Clara, CA) according to standard protocol of the manufacturer.
Scan protocol
The arrays were scanned using the GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA).
