strain: Sprague Dawley developmental stage: adult sex: male treatment group: rotenone tissue: substantia nigra pars compacta
Treatment protocol
Rotenone (Sigma, Lyon, France) was dissolved in a solution of dimethylsulfoxide (DMSO) / polyethylene glycol (PEG) (1/1) containing the non-ionic surfactant polyol Pluronic® F-127 (Interchim, Montluçon, France) used as a dispersing reagent at the final concentration of 0.05%. Prior to the implantation, Alzet® osmotic mini-pumps (2ML4, Charles River Laboratories, Saint Germain sur l’Arbresle, France) were filled with the solvent or rotenone and incubated overnight at 37°C in a sterile saline solution (NaCl 0.9%, wt/vol) according to manufacturer’s instructions. Rats were anaesthetized by an intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg). Osmotic pumps were implanted subcutaneously and diffused either the solvent (SOLV, control group, n = 3) or 0.5 mg/kg/day rotenone (ROT group, n = 3) for 28 days. The final delivery of rotenone was calculated with respect to average body weight at the time of the implantation. In order to monitor potential side effects of pesticide exposure, body weight was recorded daily for each rat throughout the experimentation.
Growth protocol
Male Sprague Dawley rats aged of 5 months were housed with ad libitum access to food and water in a temperature- (24 + 1°C) and humidity- (60%) controlled room under a 12 h / 12 h light-dark schedule (lights on at 08.00 h).
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from laser-capture microdissected rat substantia nigra pars compacta (SNpc) using the RNeasy mini-kit (QIAGEN, Courtaboeuf, France) according to manufacturer’s instructions, including the additional step of DNase treatment. Total RNA (2 ug) was amplified and labeled by a round of in vitro transcription using a MessageAmp aRNA kit (Ambion, Cambridgeshire, U.K.) following the manufacturers protocol. Before amplification, spikes of different concentrations of synthetic mRNA were added to all tubes. These positive controls were used to verify the quality of the process. Amplified RNA (aRNA) was measured with an ultraviolet spectrophotometer and the quality verified on picochips using the Agilent bioanalyzer.
Label
biotin
Label protocol
Biotin-labeled aRNA (10 ug) was fragmented with 5 uL of fragmentation buffer (GE Healthcare, Amersham, Saclay, France) in a final volume of 20 uL.
Hybridization protocol
Fragmented aRNA was added to Amersham hybridization solution (Amersham) (final volume 260 uL) and injected onto CodeLink Uniset mouse 35K bioarrays containing 35,000 mouse oligonucleotide gene probes (Amersham). The arrays were hybridized overnight at 37-C at 300 rpm on a rotary mixer in an incubator, washed at 46-C for 1 hour in stringent TNT buffer (100 mM Tris, 150 mM NaCl, 0.02% Tween 20; all from Sigma- Aldrich, Saint Quentin-Fallavier, France), incubated in 3.4 mL of streptavidin-Cy5 (Amersham) solution for 30 minutes, washed 4 times in 240 mL of TNT buffer, rinsed twice in 240 mL of water containing 0.2% Triton X-100, and dried by centrifugation at 600 rpm.
Scan protocol
The arrays were scanned with a Genepix 4000B scanner (Axon Instruments, Dipsi Industrie, Chatillon, France) using Genepix software with the laser set at 635 nm, the power at 100%, and the photomultiplier tube voltage at 60%. The scanned image files were analyzed using CodeLink expression software version 4.0, which produces both raw and normalized hybridization signals for each spot on the array.
Description
Cumulative low doses of the organic pesticide rotenone in the adult rat substantia nigra pars compacta
Data processing
The CodeLink software (version 4) normalizes the overall raw hybridization signal intensity on each array to the median of the array (median intensity is one after normalization).
