Computational protocol: Cocaine Administration and Its Withdrawal Enhance the Expression of Genes Encoding Histone-Modifying Enzymes and Histone Acetylation in the Rat Prefrontal Cortex

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Protocol publication

[…] The Rat 4x44K Gene Expression Array v2 (Agilent Technologies, USA), representing 39,000+ rat genes and transcripts, was used to assess gene expression in the rat PFC on the third day of extinction training. Sample labeling and hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol. Briefly, four pools of RNA (each including the RNA of two rats at equal concentrations) from three experimental groups (AC, YC, and YS) were prepared. A starting amount of 2 μg of total RNA was converted to complementary DNA (cDNA) and transcribed into cRNA in the presence of cyanine 3-UTP. Each complementary RNA (cRNA) sample (1.65 μg) was hybridized for 17 h at 65 °C with rotation and then washed to remove nonspecific hybridization.Microarrays were scanned using the Agilent Microarray Scanner and Feature Extraction software (v 11.0.1.1) (Agilent Technologies, USA). Data normalization and processing were carried out using the GeneSpring GX software, v. 12.1 (Agilent Technologies, USA). The scatterplot method was used to visualize variations (or reproducibility) in gene expression between arrays (Supplementary Material, Fig. ). [...] The cDNA was synthesized by reverse transcription using total RNA (1 μg) and random hexamer primers with the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, USA) following the manufacturer’s protocol.All PCR reagents were purchased from Life Technologies (USA) if not otherwise indicated. Real-time PCR analysis was performed in duplicate on a 96-well plate using the Bio-Rad CFX96 Touch™ Real-Time PCR Detection System. The 10-μl PCR reaction contained 4.5 μl of cDNA (diluted 1:2 in nuclease-free water), 5 μl of 2× TaqMan Expression Master Mix, and 0.5 μl of TaqMan™ Gene Expression Assays for ten selected genes and one endogenous control (Hprt1) (assay ID numbers used are listed in Table ). PCR cycling conditions were as follows: an initial step of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and then 60 °C for 60 s. The threshold cycle (Ct) was collected using the CFX Manager™ software. The relative expression of target genes was calculated by comparative Ct method (2−ΔΔCt). Our previous analysis showed that the transcript of Hprt1 gene was more stable than Gapdh; therefore, it served as a normalization control in this analysis. [...] The data are expressed as the means ± SEM. The behavioral data were analyzed by two-way ANOVA for repeated measures followed by a post hoc Newman-Keuls’ test using the Statistica v.10 software. The statistical analysis of molecular data was conducted using the Prism software (GraphPad Software, v. 7.0). The statistical differences between gene expression levels and Western blot data were assessed by one-way ANOVA with Dunnett’s post hoc test; significance was set at p < 0.05.R software (v. 3.1.2) was used to conduct the unpaired Student’s t test and one-way ANOVA followed by the Benjamini and Hochberg false discovery rate (FDR) for multiple comparisons for correction of microarray data. To select differentially expressed genes between the AC and YS groups, cutoffs were set as FDR ≤0.1 and values of log2 FC ≥1.0 or ≤−1.0 were used. […]

Pipeline specifications

Software tools GeneSpring GX, CFX Manager, Statistica
Applications Miscellaneous, Gene expression microarray analysis
Organisms Rattus norvegicus
Chemicals Cocaine