Microarray Analysis
Stress is associated with drug abuse and the drug craving associated with recidivism. Thus, this study investigated the functional genomics of stress and the opioid system in cellular adaptations to substance abuse. Patterns of gene expression were investigated using cDNA microarray hybridization analysis in human neuroblastoma SHSY-5Y cells. Cells were incubated in the presence of hormone-depleted serum and subsequently treated for 2-days with the glucocorticoid receptor agonist dexamethasone (30 nM); the ยต-opioid receptor agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate (DAMGO; 1 nM); and dexamethasone (30 nM) plus DAMGO (1 nM).
For the cDNA gene expression microarrays, n=3 culture dishes were tested as biological replicates, and each was tested with a duplicate technical reference. After isolation, the RNA samples were purified using the RNeasy MinElute Cleanup Kit ( Qiagen , Valencia , CA, USA ). Total RNA purity was confirmed on a 1.0% Formaldehyde/Denaturing agarose gel. cDNA was produced by reverse transcription using the SuperScript reverse transcriptase Indirect cDNA Labeling System (Invitrogen, Carlsbad , CA , USA ). A combination of equal amounts of cDNA from each of the four groups was created as the Reference Standard. The cDNA was conjugated to Alexa Fluor 555/Green (Molecular Probes Inc., Eugene , OR , USA ) for reference standard or Alexa Fluor 647/Red (Molecular Probes Inc., Eugene , OR , USA ) for treatment groups. Subsequently, the cDNA was hybridized to a spotted 10,000-oligonucleotide human gene (MWG Biotech AG, Germany ) array chip ( Pennsylvania State University College of Medicine/Milton S. Hershey Medical Center/JDRF Functional Genomics Core Facility ). Fluorescence intensities were quantitated at the following excitation/emission wavelengths: Alexa Fluor 555 - 555 nm/565 nm; Alexa Fluor 647 - 650 nm/670 nm. The sample data were normalized to the reference standard data to control for variability within samples.
Normalized data were analyzed using GeneSpring software (Agilent Technologies, Santa Clara , CA , USA ). The normalized gene probe intensities underwent Lowess normalization and gene expression filtration. The drug treatment groups were analyzed using 1-Way Analysis of Variance (ANOVA) followed with Dunnett's, Tukey's or Newman-Keuls post hoc tests to test for statistical significance. Those genes showing a statistically significant difference of 1.2-fold or greater from control were identified, and classified into ontological categories including Biological Processes and Molecular Functions.
Normalized Data Values