|
| Status |
Public on Jul 14, 2010 |
| Title |
MB3 (S14611) |
| Sample type |
RNA |
| |
|
| Source name |
Medulloblastoma, nonirradiated
|
| Organism |
Mus musculus |
| Characteristics |
strain: C3B6F1
genotype: Ptch1+/-
tissue: medulloblastoma
gender: female
age: 172
irradiation: none
|
| Treatment protocol |
Ptch1+/- mice (C3B6F1 background) were irradiated at postnatal day 1 using a Pantak HF-320 X-ray generator (Pantak Ltd., East Haven, CT, USA). Mice were observed daily until moribund and then were killed under ether anesthesia.
|
| Growth protocol |
All animals were bred under conventional conditions in our animal facility. They were housed in an air-conditioned room (23 ± 3˚C) with a relative humidity of 40 ± 10% under an alternating 12-hour light/dark schedule (lights on at 7:00am).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were purified by using AllPrep DNA/RNA mini kit (Qiagen, Valencia, CA) and Trizol Reagent (Invitrogen, Carlsbad, CA). Briefly, samples were homogenized in RLT plus buffer included in the kit. The homogenates were ten-fold diluted with Trizol Reagent and total RNA was subsequently purified according to manufacturer’s instructions for Trizol Reagent. The quality of total RNA was assessed by using the QIAxcel system (Qiagen). RNA was quantified using a NanoDrop-1000.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.8 ug RNA using the One-Color Low RNA Input Linear Amplification kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity > 10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4122F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565BA) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in medulloblastoma developed in nonirradiated mouse.
S-type
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5.1 (Agilent) using default parameters (protocol GE1-v1-v5_95_Feb07 and Grid: 014868_D_F_20080627) to obtain background-subtracted Processed Signal intensities. Data were analyzed with GeneSpring GX 10.0.2.
Expression data were normalized both “per chip” to the 75th percentile of all measurements in that sample and “per gene” to median expression levels of the gene across all samples (Median_all) or to mean expression levels of the gene of Sample 'MB1 (S14516)' (GSM480632) and Sample 'MB2 (S14610)' (GSM480633) (VALUE).
|
| |
|
| Submission date |
Dec 07, 2009 |
| Last update date |
Jul 14, 2010 |
| Contact name |
Takashi Takabatake |
| E-mail(s) |
batake@nirs.go.jp
|
| Organization name |
National Institute of Radiological Sciences
|
| Street address |
Anagawa 4-9-1, Inage-ku
|
| City |
Chiba |
| State/province |
Chiba |
| ZIP/Postal code |
263-8555 |
| Country |
Japan |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE19360 |
Integrated array-CGH and expression microarray analyses on medulloblastomas in heterozygous Ptch1 mice, expression |
| GSE19384 |
Integrated array-CGH and expression microarray analyses on medulloblastomas in heterozygous Ptch1 mice |
|
