|
| Status |
Public on Dec 15, 2006 |
| Title |
GW7845 + PolyIC 2 |
| Sample type |
RNA |
| |
|
| Source name |
primary macrophages
|
| Organism |
Mus musculus |
| Characteristics |
Strain: C57BL/6
Gender: female
Age: 6wks
Tissue: Thioglycollate-elicited macrophage
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Standard protocol from Qiagen RNeasy Mini Kit (Cat no. 74106).
|
| Label |
biotin
|
| Label protocol |
Assess the concentration and quality of the total RNA sample by Agilent Bioanalyzer.
Incubate total RNA (0.5 ug), diluited bacterial mRNA controls and T7 oligo (dT) primer for 10 min at 70 C. Add first-strand reaction components and incubate 2 hours at 42C.
Add the first-strand cDNA product to the second-strand reaction components and incubate for 2 hours at 16 C. Purify and elute twice with 30 ul EB buffer each time. Concentrate the purified cDNA to 9.5 ul per tube. 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 and elute the cRNA twice, each time with 50 ul nuclease-free water. Measure the absorbance at 260 nm and 280 nm to determine the ratio (A260:A280=2).
|
| |
|
| Hybridization protocol |
In 25 ul total volume, add 10 ug of cRNA to 5 ul of 5x fragmentation buffer and incubate at 94 C for 20 min. Bring 10 ug of fragmented cRNA, 78 ul of hybridization buffer component A, and 130 ul of hybridization buffer component B to a final volume of 260 ul with water. Incubate at 90C for 5 min and immediately chill on ice for 5 min.
Slowly inject 250 ul 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 18-24 h at 37C. 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 rhe bioarray rack to the large reagent reservoir containing preheated 0.75xTNT, and incubate at 46 C for 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 . Rinse the bioarrays in o.1xSSC/0.05%Tween for 30 sec. Immediately follow the rinse with centrifugation to dry bioarrays, and store dried bioarrays in the dark
|
| Scan protocol |
We scaned bioarrays with Agilent Scanner G2505B and analyzed with CodeLink Expression Analysis software.
|
| Description |
NA
|
| Data processing |
Data Processing: Data were normalized using a multi-loess technique described in Corbeil et al (Journal of Molecular Endocrinology (2004) 33:1–9).
|
| |
|
| Submission date |
Nov 21, 2006 |
| Last update date |
Nov 22, 2006 |
| Contact name |
Christopher Glass |
| E-mail(s) |
ckg@ucsd.edu
|
| Phone |
858-534-6011
|
| Organization name |
University of California, San Diego
|
| Department |
CMM
|
| Lab |
Glass Lab
|
| Street address |
9500 Gilman Dr.
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL1310 |
| Series (1) |
| GSE6346 |
Differential repression of TLR responses by PPARg and LXRs |
|
