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Status |
Public on Aug 30, 2015 |
Title |
cecumdead_28days_1 |
Sample type |
RNA |
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Source name |
cecum,epithelial cell,dead,28days,1
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Organism |
Mus musculus |
Characteristics |
agent: dead, 28days tissue: cecal epithelial cell gender: female age: 10-20y
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Treatment protocol |
GB mouse was prepared from GF mouse by live or dead BbrY. Cecal epithelial cells were collected 3 days or 28 days after live or dead BbrY association. Cecal epithelial cells were released by incubation with HANKS including 4 mM EDTA and 10 mM HEPES at 37 °C and collected by centrifugation after being washed with PBS and filtrated with a filter (100 mesh). The collected epithelial cells were treated with TRI-zol reagents and stored at -80 °C.
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was prepared using the Chloroform and isopropanol and DNase treatment at 37 °C. The total RNA was extracted from TRI-zol reagent buffer of cecal epithelial cells. RNA was quantified using the spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
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Label |
Cy3
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Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from total RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions. Dye incorporation and cRNA yield were checked with the Spectrophotometer.
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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 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers 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 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) and 1 minute at room temperature acetonitorile.
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Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) 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 XDRHi 100% and XDRLo 10%).
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Description |
Gene expression in ex-GF mouse 28days after dead BbrY association
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Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5 (Agilent) using default parameters (protocol GE1-v5_91 and Grid: 014868_D_20060807) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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Submission date |
May 08, 2009 |
Last update date |
Aug 30, 2015 |
Contact name |
tatsuichiro shima |
E-mail(s) |
tatsuichiro_shima@yahoo.co.jp
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Phone |
+81-42-577-8952
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Organization name |
Yakult central institute for microbiological research
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Department |
basic research
|
Lab |
intestinal function research
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Street address |
izumi5-11
|
City |
kunitachi-si |
State/province |
Tokyo |
ZIP/Postal code |
1860086 |
Country |
Japan |
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Platform ID |
GPL4134 |
Series (1) |
GSE16021 |
The difference of gene expression for live or dead BbrY associated mice. |
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