tissue: neuronal tissue transgenic: none strain: DBA2/J (DBA) Sex: female age: 12 weeks treatment: none age of treatment: none
Treatment protocol
As described in (D'Amato et al., 2011; Di Segni et al., 2016; Ventura et al., 2013), pups from the same litter spent the first postnatal day (PND0) with their biological mother. On PND1, litters were randomly assigned to experimental (RCF) or Control (Cont) treatment. RCF pups were fostered by introducing the entire litter into the home cage of a different dam whose pups had just been removed and moved to a different adoptive mother. This procedure was repeated daily (4 times, from PND1 up to PND4) until the fourth adoptive mother was reached. Pups were left with the last adoptive mother until weaning. Control litters were only picked up daily and reintroduced in their home cage, for the same period, and had their mothers returned within 30 s. Animals were weaned at PND28, separated by sex and housed in groups of 4 littermates. All experiments were performed on 3-month-old adult female mice. To prevent potential estrous cycle group synchronization, experimental subjects for cross-fostered and control female groups were sorted by collecting not >2 individuals per cage/litter (Ventura et al., 2013; Di Segni et al., 2016; 2017). Four treatment groups were used for this experiment: RCF C57, Control C57, RCF DBA, Control DBA.
Growth protocol
C57BL/6J (C57) and DBA2/J (DBA) female mice (Charles River Laboratories, Italy), 9–12-weeks-old at the time of the microarray experiments, were housed with water and food available ad libitum. Room temperature (21 ± 1 °C) and a 12:12 h light–dark cycle (lights on at 07.00 PM) were kept constant. Adequate measures were taken to minimize pain or discomfort of mice. All experiments were carried out in accordance with Italian national law (DL 116/92 and DL 26/2014) on the use of animals for research based on the European Communities Council Directives (86/609/EEC and 2010/63/UE).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from nucleus accumbens (NAc) and medial prefrontal cortex (mpFC) , with n=4 samples per treatment group (RCF C57, Control C57, RCF DBA, Control DBA). Total RNA was extracted using Trizol (Life Technologies, Inc, USA), according to the manufacturer's instructions, and DNAse treated by Qiagen columns. Quality and integrity of each sample was checked using the Agilent BioAnalyzer 2100 (Agilent RNA 6000 nano kit): samples with a RNA Integrity Number (RIN) index lower than 8.0 were discarded. All the experimental steps involving the labelling, hybridization and washing of the samples were done following the one-color Agilent protocol.
Label
Cy3
Label protocol
For each sample 200ng of Poly A+RNA were labelled, using Cyanine 3-CTP (Agilent) in separate labelling reactions, with the Agilent Low Imput Linear Amplification Kit and purified with Qiagen’s RNeasy mini spin columns.
Hybridization protocol
One-color mixes of labelled cRNA pairs were hybridized to 8x60K whole mouse genome oligonucleotide microarray (Agilent grid ID 028005), at 65°C for 17 hours using Agilent’s Gene Expression Hybridization Kit. The hybridized microarrays were disassembled at room temperature in Agilent Gene Expression Wash Buffer 1. After the disassembly, the microarrays were washed in Gene Expression Buffer 1 for one minute at room temperature, followed by washing with Gene Expression Wash Buffer 2 for one minute at 37°C. The microarrays were then treated with acetonitrile for one minute at room temperature.
Scan protocol
Post-hybridization image acquisition was accomplished using the Agilent Scanner G2564C. Data extraction from the 20 bits TIFF images was accomplished by Agilent Feature Extraction (FE) ver 10.7 software using the standard Agilent one-color gene expression extraction protocol (GE1_107_Sep09 for FE ver 10.7 ).
Description
medial prefrontal cortex tissue from DBA_mpFC_ctr mouse
Data processing
The “gProcessedSignal” data column of the Feature Extraction *.txt output file was used, containing the median signals corrected by spatial and multiplicative detrend. Data quality filtering was performed using R, discarding features with the Feature Extraction flag gIsWellAboveBG=0 in any sample, wich is a statistical method approximately equivalent to discarding features with a Signal/Noise ratio smaller than 2.0-3.0, where Signal = (median of the spot - median spot background level) and Noise is the Standard Deviation of the median spot background. Data were normalized to the 75th percentile in Log2 scale. Differentially expressed genes were selected by a combination of fold change and moderated T-test thresholds (with FDR Pvalue correction) by the R-Bioconductor tool Limma (FDR<0.05; |Log2 fold-change ratio| >1.0 equivalent to 2.0 fold in linear scale).
Role of neuronal Xlr4 gene expression in cocaine addiction vulnerability: a microarray study in medial prefrontal cortex and Nucleus Accumbens of C57 and DBA mice.
Data table header descriptions
ID_REF
VALUE
Log2 filtered expression values, normalized to the 75th percentile.
