Timed pregnant Swiss Webster dams(CFW; Charles RiverLab. Wilmington, MA)
Treatment protocol
Timed pregnant Swiss Webster (CFW; Charles River Lab. Wilmington, MA) dams were maintained in individual cages in a climate-controlled room on a 12/12-h light/ dark cycle. They were divided into two groups. The first (experimental) group received subcutaneous (at the dorsum of the neck) injections of 20 mg/kg cocaine hydrochloride (Research Technology Branch, National Institute of Drug Abuse, Rockville, MD) dissolved in 200 mkl of 0.9% saline, twice a day (at 8:00 AM and 8:00 PM) from 8th through 18th day of pregnancy (E8–E18). The cocaine treatment was designed to replicate the one capable of reducing cerebral cortical mass in mouse offspring. Also, the relatively protracted period the chronic treatment was chosen to maximize the changes in the tissue expression of cocaine-regulated genes of interest. Throughout the treatment, all mice were weighed daily, and, from E8, the control and experimental animals were pair-fed, with the daily amount of food (Mouse Chow; Ralston Purina Saint Louis, MO) provided to each control dam being matched to that consumed by the paired experimental dam. Water was available ad libitum. We found that this feeding regiment resulted in similar weight gains from E8 to E18 in both experimental and control animal groups (46.41F0.45% and 44.99F0.59% respectively). After delivery animals were grown one month. On P30 the animals were anesthetized by peritoneal injection of 50 mg/kg sodium pentobarbital (Abbott Lab., Abbott Park, IL). The frontal cerebral wall (containing cerebral cortex and underlying transient cortical zones anterior to the striatal level) and the occipital cerebral wall (containing cerebral cortex and underlying transient cortical zones posterior to the level of the hippocampus) were dissected and stored in liquid nitrogen prior to analysis. The animal experimentations used in this study were approved by the University of Maryland Animal Care and Use Committee.
Extracted molecule
total RNA
Extraction protocol
Tissue from control and experimental animals were used in the processing of a single microarray slide (Part G4121A, Agilent Technol). Five slides were processed per region. For a given region, the tissue for each microarray slide was obtained from a separate set of control and experimental litters. Total RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA). The yield of total RNA was determined by absorbance at 260 nm on a DU 640 Spectrophotometer (Beckman Coulter, Fullerton, CA). The 260/280 nm ratios of the samples were >1.8.
Label
Alexa647
Label protocol
For each assay, 5 mkg of RNA from control tissue and 5 mkg of RNA from experimental tissue were reverse transcribed using 3DNA Array Detection Kit (Genesphere, Hatfield, PA) with primers containing different dcaptureT sequences for control and experimental samples. SuperScript II Reverse Transcriptase for these reactions was purchased from Gibco BRL (Gaithersburg, MD). For fluorescent labeling of microarray-bound cDNA, the slides were incubated for 3 h at 65 8C with Capture Reagent Hybridization Mixture from 3DNA Array Detection Kit (Genesphere).
Timed pregnant Swiss Webster dams(CFW; Charles RiverLab. Wilmington, MA)
Treatment protocol
The second (control) group was subjected to the same schedule of 0.9% saline only injections.
Extracted molecule
total RNA
Extraction protocol
the same as Channel 1
Label
Alexa 546
Label protocol
The same as Channel 1, but with Alexa 546
Hybridization protocol
The resultant cDNAs were hybridized to microarray slides for 16 h in Agilent Hybridization Buffer (Agilent Technol) at 60C. Upon completion of the hybridization, the slides were washed for 10 min with 0.005% Triton in 6xsodium chloride/sodium citrate buffer (pH 7; SSC) at room temperature and then for five more min with 0.005% Triton in 0.1xSSC and 1 more minute in 0.1xSSC (pH 7), both at 4C. Washed slides were air-dried for 30 s. For fluorescent labeling of microarray-bound cDNA, the slides were incubated for 3 h at 65C with Capture Reagent Hybridization Mixture from 3DNA Array Detection Kit (Genesphere). In this mixture, Alexa 546 fluorochromei ncorporating dendrimers contained single-stranded arms complimentary to the dcaptureT sequences used in the reverse transcription of RNA from control tissue, while Alexa 647 fluorochrome-incorporating dendrimers contained arms complimentary to the capture sequences used in reverse transcription of RNA from experimental tissue. The labeling reaction was terminated by washing of the microarray slides at room temperature for 10 min with 0.005% Triton in 2xSSC and for another 10 min with in 0.2xSSC. After that, the slides were air-dried for 30 s.
Scan protocol
The processed microarray slides were scanned at 10 mkm resolution on a GenePix 4100A scanner (Axon Instr., Union City, CA) with the laser excitation at 532 nm [emission filter 575DF35 (green); photomultiplier voltage 550] for Alexa 546 (control samples) and the laser excitation at 635 nm [emission filter 670DF40 (red); photomultiplier voltage 695] for Alexa 647 (experimental samples). The signals were converted into 16-bits-per pixel resolution images, providing color depth of 65,536 levels. The densitometry was performed with GenePix Pro 4.1 software (Axon Instr.). Background was subtracted using the dlocal background correctionT procedure available in the software. Quality control utilized 255 positive controls, 161 negative controls and 646 QC-spots present on Agilent’s arrays.
Description
Pregnant Swiss Webster mice were used as a model because cocaine treatment in these animals has been shown to result in offspring with reduced cerebral cortical mass.
Data processing
For replicates within a slide, median signal values were calculated. For each array, the data were normalized in Acuity 3.1 software (Axon Instr.) by applying locally weighted scatterplot smoothing (LOWESS) transformation. The between array scale normalization was done using intensity log10 ratio distribution box plots (GeneSight software, BioDiscovery, El Segundo, CA) for verification. The box plots of the fully normalized data showed fairly similar spread for all microarray slides used in this study. Statistical analysis of the normalized data was conducted by the Significant Analyses for Microarray Algorithm (SAM) using Excel macros available at Stanford SAM website. In the analyses of both frontal and occipital regions of the fetal cerebral wall, the cut-off thresholds were set to identify the maximum number of cocaine exposure-regulated genes at the minimal false discovery rate (FDR) allowed by SAM for a given set of microarray data. For both regions, the FDR rates were <0.5%.
