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| Status |
Public on Oct 28, 2013 |
| Title |
Total Homogenate_Alcohol_8 |
| Sample type |
RNA |
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| Source name |
Total Homogenates (the standard prep) from extended amygdala
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| Organism |
Mus musculus |
| Characteristics |
background strain: C57Bl6/J
age: 2 months
gender: female
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| Treatment protocol |
Mice were given a 24-hour/day access to a two-bottle choice protocol using bottles containing water and/or 20% ethanol and the volume consumed from both bottles was recorded daily as described by (Osterndorff-Kahanek, Ponomarev et al. 2013). Bottle positions were changed daily to control for position preferences. All procedures were approved by the Institutional Animal Care and Use Committee and adhere to NIH Guidelines. The brains were removed and washed for one minute with 1ml of ice-cold-Homogenizing Buffer (HB) containing Hepes [20mM], EDTA [1mM](pH 7.4), RNAseOut, phosphatase and protease inhibitors (from Invitrogen, Sigma, Roche, respectively). Brains were then placed in a coronal Zivic-mouse-brain-slicer (previously washed with HB) with a 0.5mm resolution (Zivic Instruments, PA) and sliced in the following coordinates in order to isolate extended amygdala-containing slices: coronal level 56-66 [Bregma (-0.18)-(-1.155)] and 66-80 [Bregma (-1.155)-(-2.55)]. The extended amygdala was dissected and placed in a tube containing ice-cold HB (250) and homogenized using a VWR homogenizer. To minimize homogenate loss, the pestles were washed with 50ml HB after use and the wash was collected and added to the sample. 10% of the homogenate (30ml) was snap frozen in liquid nitrogen and stored at -80°C for RNA total homogenate (TH) analysis. The rest of the homogenate (270ml) was filtered with a 100um-pore filter and a 5um-pore filter (Millipore) (filters were washed with HB before use for protection from RNAse). To maximize yield, the filters were washed with 50ml HB after use and the wash was collected and added. The homogenate was then centrifuged at 14,000g for 20 minutes at 4°C in order to pellet the cell fraction containing SN (Quinlan, Philpot et al. 1999, Raab-Graham, Haddick et al. 2006, Sosanya, Huang et al. 2013). The supernatant was removed and the pellet snap frozen and stored at -80°C for SN RNA analysis.
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| Growth protocol |
Adult (two month old) C57BL/6J female mice were maintained at the University of Texas at Austin Animal Research Center. Mice were given a one-week acclimation period in combined housing and another week to acclimate to the bottle position in individual housing. Food and water were provided ad libitum and monitored daily, as were the temperature and light/dark cycles.
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| Extracted molecule |
total RNA |
| Extraction protocol |
We extracted total RNA from the extended amygdala of 8 alcohol treated mice and 13 controls and 42 microarrays were used for the alcohol-control analysis, 21 SN and 21 paired TH. Total RNA was extracted from the SN and TH with the Direct-Zol RNA extraction kit (Zymo, Japan), using the small IC extraction columns according to manufacturer’s instructions. The RNA was quantified using nanodrop (NanoDrop 1000, Thermo Fisher Scientific Inc., IL) and assayed for quality using Agilent 2100 Tape Station (Agilent Technologies, CA). The cut-off criteria were set on 280/260 > 1.7, RIN > 6.5, and amount of total RNA > 500ng. We used two validation methods for the preparation: flow cytometer analysis of SN filtration according to (Gylys, Fein et al. 2004, Sokolow, Henkins et al. 2012) and immunocytochemistry (Gylys, Fein et al. 2004, Williams, Mehrian Shai et al. 2009).
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| Label |
biotin
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| Label protocol |
RNA samples were processed at the University of Texas Southwestern Medical Center microarray facility in Dallas. mRNA was amplified and biotin-labeled using the Illumina TotalPrep RNA Amplification kit (Ambion, TX) and hybridized to Mouse WG-6 v2.0 Expression BeadChips (Illumina, CA). Each array contained SN and paired TH samples from control and alcohol-treated mice. These were assigned randomly to each array.
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| Hybridization protocol |
According to manufacturer’s instructions.
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| Scan protocol |
According to manufacturer’s instructions.
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| Description |
TH15
Alcohol treated mice were given a 24-hour access for 30 days to a two-bottle choice, using a bottle containing water and a bottle containing 20% ethanol
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| Data processing |
The data were preprocessed using variance stabilization transformation (variance within array) (Dunning, Ritchie et al. 2008, Lin, Du et al. 2008), quantile normalized (variance between arrays), and background subtracted using ‘Lumi’ package (Du, Kibbe et al. 2007, Du, Kibbe et al. 2008). Quality measures were taken before and after preprocessing using the array quality metrics package (Kauffmann, Gentleman et al. 2009, Kauffmann and Huber 2010) to remove outliers based on at least two out of the three tests in the package and taking care that the normalization did not skew the data (only two TH samples, out of 42 total arrays, failed to pass the cut off and so were removed from the analysis). This package was also used to generate the Principle component analysis. We determined the transcripts detected on 80% of the arrays with a p < 0.05.
The data tables contain Illumina dentifiers, raw or normalized (scaled) signal count and detection p-values. Each normalized sheet contains data that was normalized using a specific group of samples. The normalized data is presented for the transcripts that had a detection p-value < 0.05 for atleast 80% of the samples.
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| Submission date |
Oct 25, 2013 |
| Last update date |
Oct 28, 2013 |
| Contact name |
Dana Moat |
| E-mail(s) |
danamost@gmail.com
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| Organization name |
University of Texas at Austin
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| Department |
Waggoner center for alcohol and addiction research
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| Lab |
Harris and Mayfield
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| Street address |
2500 Speedway, MBB 1.138
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| City |
Austin |
| State/province |
Texas |
| ZIP/Postal code |
78712 |
| Country |
USA |
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| Platform ID |
GPL6887 |
| Series (1) |
| GSE51730 |
Profiling the transcriptome: synaptoneurosomes capture the molecular effects of alcohol consumption |
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| Supplementary data files not provided |
| Processed data are available on Series record |
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