gender: Male age: P7 brain region: Amygdala treatment condition: Paroxetine
Treatment protocol
Adult females received paroxetine (10mg/kg/day) through their drinking water (or normal drinking water in the case of controls) beginning one week prior to conception, throughout gestation, until the offspring were weaned on postnatal day (P)21.
Growth protocol
Standard rat housing conditions
Extracted molecule
total RNA
Extraction protocol
RNA was isolated and purified using the TRIzol method. Briefly, tissue was homogenized in TRIzol (Life Technologies) and incubated at room temperature to allow dissociation of nucleoproteins. Chloroform was then added to each homogenized sample and centrifuged at 12,000g for 15 min at 4°C. Next, RNA was precipitated with isopropyl alcohol and spun down at 12,000g for 10 min at 4°C. The pellet was washed in ethanol, allowed to air dry, and resuspended in 100µl DEPC-H2O. RNA quantity and quality were measured by NanoDrop ND-1000; RNA integrity was assessed by standard denaturing agarose gel electrophoresis.
Label
Cy3
Label protocol
Total RNA from each sample was linearly amplified with Agilent's Low Input Quick Amp Kit (Agilent Technology). Double-stranded cDNA (ds-cDNA) was synthesized from the amplified cRNA using an Invitrogen SuperScript ds-cDNA synthesis kit in the presence of 100pmol oligo dT primers. ds-cDNA was cleaned and labeled in accordance with the NimbleGen Gene Expression Analysis protocol (NimbleGen Systems, Inc., USA). Briefly, ds-cDNA was incubated with 4μg RNase A at 37°C for 10 min and cleaned using phenol:chloroform:isoamyl alcohol, followed by ice-cold absolute ethanol precipitation. The purified cDNA was quantified using a NanoDrop ND-1000. For Cy3 labeling of cDNA, the NimbleGen One-Color DNA labeling kit was used according to the manufacturer's guideline detailed in the Gene Expression Analysis protocol (NimbleGen Systems, Inc., Madison, WI, USA). One microgram of ds-cDNA was incubated for 10 min at 98°C with 1 OD of Cy3-9mer primer. Then, 100pmol of deoxynucleoside triphosphates and 100U of the Klenow fragment (New England Biolabs, USA) were added and the mix incubated at 37°C for 2 hours. The reaction was stopped by adding 0.1 volume of 0.5M EDTA, and the labeled ds-cDNA was purified by isopropanol/ethanol precipitation.
Hybridization protocol
Microarrays were hybridized at 42°C 16-20h with 4μg of Cy3 labeled ds-cDNA in NimbleGen hybridization buffer/hybridization component A in a hybridization chamber (Hybridization System - NimbleGen Systems, Inc., Madison, WI, USA). Following hybridization, washing was performed using the NimbleGen Wash Buffer kit (NimbleGen Systems, Inc., Madison, WI, USA).
Scan protocol
After being washed in an ozone-free environment, the slides were scanned using the Axon GenePix 4000B microarray scanner. Slides were scanned at 5μm/pixel resolution using an Axon GenePix 4000B scanner (Molecular Devices Corporation) piloted by GenePix Pro 6.0 software (Axon). Scanned images (TIFF format) were imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level files and Gene level files were generated after normalization.
Data processing
All gene level files were imported into Agilent GeneSpring GX software (version 12.6) for further analysis. Probes with a raw expression value less than 50 for any sample were excluded from the analysis. The samples were then filtered to exclude any probes designated “predicted mRNA model”, as determined by the most recent RefSeq database annotations. Following experimental grouping (i.e., perinatal SSRI- versus vehicle-exposed LR at a particular developmental time point and specific brain region), differentially expressed genes with statistical significance were identified through volcano plot filtering, using cutoffs of fold change >1.5, p-value <0.05 [Bonferroni family-wise error rate (FWER)].
