|
| Status |
Public on Aug 22, 2011 |
| Title |
Hypothalamus_KISS1-/-_mutant_rep4 |
| Sample type |
RNA |
| |
|
| Source name |
KKO biological rep 4, Affy processing batch 3
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129S6/Sv/Ev
genotype: KISS1-/- mutant
gender: male
|
| Treatment protocol |
Not applicable.
|
| Growth protocol |
Microdissected hypothalamic tissue from KISS1-/- mutant, GPR54-/- mutant, and wild type mice. Male 129S6/Sv/Ev wildtype, 129S6/Sv/Ev Gpr54- or 129S6/Sv/Ev Kiss1- knockout mice were housed under conditions of 12 hours of light with ad libitum access to food and water.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After removal of the brain, the meninges and optic chiasm were discarded and the hypothalamus was isolated. The external limits for this dissection were: lateral, the external border of the medial preoptic area and more caudally, the lateral borders of the mammillary nucleus; dorsal, 1.5 mm depth; anterior, the anterior limit of the nucleus of the vertical limb of the diagonal band (bregma + 1.34 mm); and posterior, the posterior limit of the mammillary nucleus (bregma – 3.40 mm). The whole hypothalamus contained at least the following main hypothalamic nuclei: arcuate nucleus (ARC), anteroventral periventricular nucleus (AVPV), anterodorsal preoptic nucleus (ADP), magnocellular nucleus (MA), supraoptic (SON) and paraventricular (PVN) nuclei, medial preoptic area (MPO) and the diagonal band of Broca (DBB). Immediately after dissection, hypothalami samples were placed in 600 μl 21 of RNAlater (Applied Biosystems, UK) and kept at 4°C for 24 h and then stored at -20°C until RNA extraction.
|
| Label |
biotin
|
| Label protocol |
5.5 μg of single stranded cDNA was fragmented with Uracil DNA Glycosylase (UDG) and APE 1 then labeled with biotin allonamide triphosphate (TdT) using the GeneChip Whole Transcript Terminal Labeling Kit before being hybridized.
|
| |
|
| Hybridization protocol |
Hybridized for 17 h to the Mouse 1.0 ST Array chip in the GeneChip Hybridization Oven 640 using the GeneChip Hybridization, Wash and Stain Kit.
|
| Scan protocol |
Before scanning the chip on the GeneChip Scanner 3000 7G, each chip was run through the GeneChip Fluidics Station 450 for washing and staining with Streptavidin Phycoerythrin (SAPE). When the sample yielded a concentration less than required for subsequent steps in the protocol, the process was started over again and resulting volumes amalgamated (and in some instances vacuum centrifuged to increase the concentration) before moving forward to the next step.
|
| Description |
KKOMH KB15
|
| Data processing |
CEL files were processed using ArrayAssist 5.0. Raw intensity calls were normalized using quantile normalization (Bolstad, 2003) and probeset summarization undertaken with gc-rma (Wu, 2004). Outliers and probesets with greater than 1.5 fold change between genotypes and level of significance p < 0.05 were selected for further analysis by quantitative PCR (Q-PCR). A complete data matrix of ArrayAssist 5.0 quantile normalized data not provided due to the following reason: "We had a license for ArrayAssist back in 2007, and unfortunately at that time I did not save complete matrix output. The files from the Affy Exon chips were so large (1.6 million rows of data for probeset level exon data) that no software we had could process or display the data for our biologists - only filtered lists of significant results were manageable outside of software such as ArrayAssist. We no longer have an ArrayAssist license, so I am unable to generate the complete data matrix. We now have computers capable of handling larger sets of data and R/Bioconductor software for processing. I suspect that anyone wanting to use this data would not use ArrayAssist 5.0 or the older analytical methods implemented therein, but will want to operate directly with the CEL files."
Significant genes list can be found on GSE28383.xls file.
probe group file: ArrayAssist 5.0 internal affy pgf
meta-probeset file: ArrayAssist 5.0 internal affy mps
|
| |
|
| Submission date |
Apr 05, 2011 |
| Last update date |
Aug 22, 2011 |
| Contact name |
Steven McKinney |
| E-mail(s) |
smckinney@bccrc.ca
|
| Phone |
604-675-8000
|
| Organization name |
British Columbia Cancer Agency
|
| Department |
Molecular Oncology
|
| Lab |
Samuel Aparicio
|
| Street address |
675 West 10th Ave
|
| City |
Vancouver |
| State/province |
BC |
| ZIP/Postal code |
V5Z 1L3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6193 |
| Series (1) |
| GSE28383 |
Testosterone-dependent and independent transcriptional networks in the hypothalamus of Gpr54 and Kiss1 knockout male mice |
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