Total RNA containing the small RNA fraction was isolated from 100mg fresh frozen tissue from the nucleus accumbens (NAc) using the mirVana-PARIS kit (Life Technologies, Carlsbad, CA) following manufacturer's protocols. RNA concentration was measured using the Quant-iT Broad Range RNA Assay kit (Life Technologies) and the RNA Integrity Number (RIN) was measured on the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA).
Label
biotin
Label protocol
Total RNA (500ng) from each specimen was labeled using the FlashTag™ Biotin HSR RNA labeling kit (Affymetrix). Each RNA sample was spiked with 5 different oligonucleotides as positive endogenous controls to assess the efficiency of the labeling reaction. The RNA samples were subjected to a brief Poly(A) tailing reaction followed by ligation of a biotinylated signal molecule.
Hybridization protocol
Each labeled sample was hybridized onto a GeneChip® miRNA 3.0 Array.
Scan protocol
Each array was scanned on the Affymetrix GeneChip® Scanner 3000 7G (Affymetrix) and raw probe intensities stored in .CEL files by the GeneChip® Operating Software (GCOS v1.4).
Data processing
Array quality was assessed by monitoring the 3′/5′ ratios of GAPDH and by the percentage of “Present” genes (%P) and array exhibiting GAPDH 3′/5′ < 3.0 and %P > 40% were considered of good quality. Expression values were calculated following the pre-processing procedure: 1) GCRMA background correction, 2) log2 transformation, 3) quantile normalization and 4) median-polish probeset summarization using Partek Genomics Suite (PGS) v6.23 (PGS; Partek Inc., St. Louis, MO). The batch effect removal option in PGS was used to control for batch effect. mRNA and miRNA microarray quality was further assessed using a principal components analysis (PCA) which was conducted on the expression values for both array types in which samples were plotted along the first three principle components (PCs) to identify potential microarray outliers.
