|
| Status |
Public on Mar 01, 2016 |
| Title |
Nac control offspring, biological replicate 4 |
| Sample type |
RNA |
| |
|
| Source name |
C57BL/6 Nucleus Accumbens - frozen brain tissue - CONTROL
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6N
prenatal exposure: control
age: 12 weeks
tissue: brain
tissue region: Nucleus Accumbens (NAc)
|
| Treatment protocol |
CC57BL/6 mice were treated with the synthetic viral mimetic poly(I:C) (5 mg/kg, i.v.) or control (saline, i.v.) solution on gestation day 17. Offspring were subjected to cognitive and behavioral testing in adulthood, and then whole genome gene expression analysis with Affymetrix Microarray and subsequent q-PCR validation were performed on the nucleus accumbens.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
[Tissue extraction protocol] The brains from adult offspring (12 weeks) were rapidly extracted from the skull (within < 20 s) and placed on an ice-chilled plate. This was followed by preparing 1-mm coronal brain sections using razorblade cuts and subsequent micro-dissection of the brain areas of interest. We dissected the nucleus accumbens (NAc, including core and shell subregions; bregma +1.5 to +0.5 mm). Brain specimens were collected in 96-well microtiter plates kept on dry ice and allowed to freeze before storage at −80°C until further use. Total DNA and RNA were isolated using the Qiagen AllPrep DNA and RNA Mini ki. Genome-wide gene expression analyses were performed using Affymetrix microarray assays (Mouse Gene 1.1 ST Array Strips on GeneAtlas platform), following the 3’IVT one cycle labelling
Total DNA and RNA were isolated using the Qiagen AllPrep DNA and RNA Mini kit following the manufacturers' instructions
|
| Label |
biotin
|
| Label protocol |
Gene expression microarray assays were performed using Mouse Gene 1.1 ST Array Strips on GeneAtlas platform (Affymetrix), following the 3’IVT one cycle labeling and amplification protocol described in the Affymetrix GeneChip Expression Analysis Technical Manuals and in the GeneAtlas™ WT Expression Kit User Manual. In detail, to synthesize First-Strand cDNA, 250ng RNA were reverse-transcribed with the Gene Atlas 3’IVT Express Kit or WT Expression Kit (Affymetrix, Santa Clara, CA, USA) using T7 oligo(dT) primer. Second-Strand cDNA synthesis was carried out using DNA polymerase and RNase H to simultaneously degrade RNA and synthesize second-strand cDNA. This step was followed by the in vitro transcription using IVT Labelling Master Mix to generate multiple copies of biotin-modified antisense-RNA (aRNA) from the double-stranded cDNA templates. Subsequently strand DNA was purified to remove unincorporated NTPs, salts, enzymes and inorganic phosphate.
|
| |
|
| Hybridization protocol |
Labelled cDNA (10ug) was then fragmented and 5.5ug were hybridized onto Mouse Gene 1.1 ST Array Strips. The reactions of hybridation, fluidics and imaging were performed on the Affymetrix Gene Atlas instrument according to the manufacturer’s protocol.
|
| Scan protocol |
GeneChips were scanned using the GeneAtlas Platform following the manufacturers' instructions.
|
| Description |
Gene expression data in control offspring
sample 7 con nac
|
| Data processing |
Affymetrix CEL files were imported into Partek Genomics Suite version 6.6 for data visualization and statistical testing. Quality control assessment was performed using Partek Genomic Suite 6.6. All samples passed the criteria for hybridization controls, labelling controls and 3’/5’ Metrics. Background correction was conducted using Robust Multi-strip Average (RMA) (Irizarry et al, 2003) to remove noise from auto fluorescence. After background correction, normalization was conducted using Quantiles normalization (Bolstad et al, 2003) to normalize the distribution of probe intensities among different microarray chips. Subsequently, a summarization step was conducted using a linear median polish algorithm (Tukey, 1977) to integrate probe intensities in order to compute the expression levels for each gene transcript. Upon data upload, pre-processing of CEL data for the complete data set [total of twelve samples; six biological replicates per sample for vehicle, six biological replicates for GD17 Poly(I:C)] was performed using ANOVA to assess treatment effects.
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| |
|
| Submission date |
Feb 16, 2016 |
| Last update date |
Mar 01, 2016 |
| Contact name |
Juliet Richetto |
| E-mail(s) |
juliet.richetto@uzh.ch
|
| Organization name |
University of Zurich - Vetsuisse
|
| Department |
Institute for Pharmacology and Toxicology
|
| Street address |
Winterthurerstrasse 260
|
| City |
Zurich |
| ZIP/Postal code |
8057 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL11533 |
| Series (2) |
| GSE77971 |
Expression data from adult mouse Nucleus Accumbens following prenatal infection |
| GSE77973 |
Expression data from adult mouse brain regions following prenatal infection |
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