|
| Status |
Public on Oct 08, 2014 |
| Title |
ICAM-1-/- DM (DM-KO) |
| Sample type |
RNA |
| |
|
| Source name |
kidney (renal cortex)
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype/variation: ICAM-1-/-
gender: male
tissue: Kidney cortex
age: 20 weeks of age
treatment group: 12 weeks after peritoneal injection of streptozotocin
|
| Biomaterial provider |
ICAM-1-/- mice (C57BL/6J background) were purchased from The Jackson Laboratory (Bar Harbor, ME).
|
| Treatment protocol |
ICAM-1-/- mice aged 8 weeks (n=5) were received an intraperitoneal injection of streptozotocin (STZ) at 200mg/kg in citrate buffer (pH 4.5).
|
| Growth protocol |
All mice had free access to standard diet and tap water. All procedures were performed according to the Guidelines for Animal Experiments at Okayama University Medical School, Japanese Government Animal Protection and Management Law (no. 105), and Japanese Government Notification on Feeding and Safekeeping of Animals (no. 6).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
12 weeks after the injection of STZ, all mice were killed, and the kidneys were harvested. Total RNA was extracted from each specimen of renal cortex using standard protocol from RNeasy midi kit (Qiagen, Valencia, CA).
|
| Label |
biotin
|
| Label protocol |
The concentration and the quality of the total RNA sample were assessed by Agilent Bioanalyzer. Biotin-labeled target cRNA was prepared using First and Second strand cDNA Synthesis Kit (Amersham Biosciences, No. 320000) and In Vitro Transcription Kit (Amersham Biosciences, No.320001). Incubate total RNA (5 μg), diluted bacterial mRNA spike controls and T7 oligo (dT) primer for 10 min at 70 C. Add first-strand reaction components and incubate 1 hour at 42C. Add the first-strand cDNA product to the second-strand reaction components and incubate for 2 hours at 16 C. Double-strand cDNA was purified using QIAquick purification kit (Qiagen), eluted twice, each time with 30μl of nuclease-free water then dried in a SpeedVac concentrator. Prepare IVT mix of biotinylated UTP, ribonucleotides, and the 10x T7 enzyme mix and add to the resuspended cDNA. Incubate at 37C for 14 hours. Purify cRNA using RNeasy Mini Kit (Qiagen) and elute the cRNA twice, each time with 50 μl nuclease-free water. Measure the absorbance at 260 nm and 280 nm to determine the ratio (A260:A280=2).
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| |
|
| Hybridization protocol |
In 25 μl total volume, add 10 μg of cRNA to 5 μl of 5x fragmentation buffer and incubate at 94 C for 20 min. Bring 10 μg of fragmented cRNA, 78 μl of hybridization buffer component A, and 130 μl of hybridization buffer component B to a final volume of 260 μl with water. Incubate at 90C for 5 min and immediately chill on ice for 5 min.
Slowly inject 250 μl of hybridization reaction mixture into array input port and seal ports with sealing strips. Set the shaker speed to 300 rpm and incubate slides for 18 hours at 37C in an Innove 4080 shaker. Remove the Flex Chamber using the hybridization removal tool. Then, place the bioarrays into the bioarray rack while it sits inside the medium reagent reservoir containing 0.75xTNT. Transfer the bioarray rack to the large reagent reservoir containing preheated 0.75xTNT, and incubate at 46 C for exactly 1 hour. Fill each slot of the small reagent reservoir with 3.4 ml of Cy5-Streptavidin working solution. Transfer the bioarray rack from the large reagent reservoir in to the small reagent reservoir and incubate bioarrays at RT for 30 min. Wash the bioarrays four times with 1xTNT at RT for 5 min. Rinse the bioarrays in 0.1xSSC/0.05%Tween by moving rack up and down 5 times in 5 seconds. Immediately follow the rinse with centrifugation to dry bioarrays, and store dried bioarrays in the dark.
|
| Scan protocol |
We scanned bioarrays with Axon GenePix 4000B and analyzed with CodeLink Expression Analysis software version 2.3.2.
|
| Description |
N/A
|
| Data processing |
The 10,000 spot intensities on the scanned microarray image were normalized to a median value of 1 and data were exported for analysis with CodeLink Expression Analysis software version 2.3.2.
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| |
|
| Submission date |
Mar 29, 2010 |
| Last update date |
Oct 08, 2014 |
| Contact name |
Satoshi Miyamoto |
| Organization name |
Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical sciences
|
| Street address |
2-5-1 Shikata-cho
|
| City |
Okayama |
| ZIP/Postal code |
700-8558 |
| Country |
Japan |
| |
|
| Platform ID |
GPL1310 |
| Series (1) |
| GSE21390 |
Gene expression analysis of diabetic kidney cortices obtained from ICAM-1 deficient mice and wild type mice. |
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