mRNA microarray experiments were performed on NAc tissue from a subset of heroin (N=22; 19 males and 3 females) and control (N=27; 22 males and 5 females) subjects at the Purdue Pharma, L.P. (Cranbury, NJ). Total RNA isolation, assessment of RNA integrity, and microarray hybridization were performed as previously described.(36) Expression data were collected using HG-U133A Affymetrix Chips (Affymetrix, Santa Clara, CA). Raw data were normalized using the Robust Multichip Average (RMA)(37) from the Affymetrix Expression Console. To control for RNA quality, actin 3′/5′ ratios of less than 3 and % present calls of 50% or greater were used. To identify Differentially Expressed Genes (DEGs), two-tailed t-tests were performed with a multiple-test correction (p<0.05) (38). Hierarchical clustering was performed on log2 expression levels of DEGs with Euclidian distance and average-linkage for constructing dendograms. Common transcription factors for DEGs were identified with Lists2Networks, a web-based software system for performing gene list enrichment analysis.(39) The transcription factor gene set library in List2Networks was created from putative targets of transcription factors.(40) All other computational analyses were performed in MATLAB. (Mathworks, Natick, MA)
