6-OHDA lesioning. Unilateral lesions were performed under anesthesia with chloral hydrate (400 mg/kg, i.p.). After immobilization on a stereotaxic frame (model 940; David Kopf Instruments), a hole was drilled in the skull for injections of 6-OHDA in the medial forebrain bundle (MFB). 6-OHDA (2 µg/µl x 5 µl in 0.9% NaCl containing 0.2 mg/ml ascorbic acid) was unilaterally injected into the left medial forebrain bundle (-4.4 mm AP, 1.2 mm ML relative to bregma and 8.4 mm below skull) over 4 minutes. At the end of each injection, the micropipette was left in place for an additional 5 min and then withdrawn slowly to prevent reflux of the solution.
Growth protocol
Animals. Male Sprague-Dawley rats (Charles Rivers Laboratories, Raleigh, NC), weighing g at the beginning of the experiments were used in the present study. Animals were housed in a humidity- and temperature-controlled room and were given free access to food and water. All animal procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee.
Extracted molecule
total RNA
Extraction protocol
METH treatment and tissue collection. One week after measuring METH-induced rotation, the animals were divided into two groups based on METH-induced rotation. The two matched groups of animals were injected with either saline or METH (2.5 mg/kg, intraperitoneally) and then sacrificed two hours after the injection. Additional control animals that had not gotten 6-OHDA injections were also used. Their brains were quickly removed, tissues were dissected on ice, snap frozen on dry ice, and stored at -80°C until used in HPLC, microarray, and quantitative PCR experiments. The experimental groups were: saline-treated controls (SC), METH-treated controls (MC), non-lesioned side of saline-treated 6-OHDA-injected rats (SNL), lesioned side of saline-treated 6-OHDA-injected rats (SL), non-lesioned side of METH-treated 6-OHDA-injected animals (MNL), and lesioned side of METH-treated 6-OHDA-injected animals (ML). HPLC. For monoamine analysis, the brain regions were homogenized in 0.01M HClO4 and centrifuged at 14, 000 x g for 15 min. DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) levels were analyzed in the brain tissue extracts using HPLC with electrochemical detector (Krasnova et al., 2007). RNA extraction. Total RNA was isolated using Qiagen RNeasy Midi kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA) and showed no degradation.
Label
Cyanine3-streptavidin
Label protocol
Brains were quickly removed, tissues were dissected on ice, snap frozen on dry ice, and stored at -80°C until used in HPLC, microarray, and quantitative PCR experiments. The experimental groups were labelled as: saline-treated controls (SC), METH-treated controls (MC), non-lesioned side of saline-treated 6-OHDA-injected rats (SNL), lesioned side of saline-treated 6-OHDA-injected rats (SL), non-lesioned side of METH-treated 6-OHDA-injected animals (MNL), and lesioned side of METH-treated 6-OHDA-injected animals (ML).
Hybridization protocol
Microarray hybridization. Microarray hybridization was carried out using Illumina’s RatRef-12 Expression BeadChips arrays (22, 523 probes) (Illumina Inc., San Diego, CA). In brief, a 600 ng aliquot of total RNA from each striatal sample was amplified using Ambion’s Illumina RNA Amplification kit (cat. no. IL1791; Ambion, Austin, TX). Single-stranded RNA (cRNA) was generated and labeled by incorporating biotin-16-UTP (Roche Diagnostics GmbH, Mannheim, Germany, cat. no. 11388908910). 750 ng of each cRNA sample were hybridized to Illumina arrays at 55 oC overnight according to the Illumina Whole-Genome Gene Expression Protocol for BeadStation (Illumina Inc., San Diego, CA, cat. # 11201828).
Scan protocol
Hybridized biotinylated cRNA was detected with Cyanine3-streptavidin (Amersham Biosciences, Piscataway, NJ, cat. #146065) and quantified using Illumina's BeadStation 500GX Genetic Analysis Systems scanner.
Description
SAMPLE 6
Data processing
The microarray data reported in the manuscript are in accordance with MIAME guidelines. The Illumina BeadStudio software was used to measure fluorescent hybridization signals. Data were extracted by BeadStudio (Illumina, San Diego, CA) and then analyzed using GeneSpring software v. 7.3.1 (Silicon Genetics, Redwood City, CA, USA). Raw data were imported into GeneSpring and normalized using global normalization. The normalized data were used to identify changes in gene expression in these 4 group comparisons. A gene was identified as changed if it showed increased or decreased expression according to an arbitrary cut-off of 1.7-fold changes at p < 0.025. In previous studies, genes identified by similar criteria were consistently validated by quantitative PCR (Krasnova et al., 2008; Cadet et al., 2009; Jayanthi et al., 2009).
