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Sample GSM146845 Query DataSets for GSM146845
Status Public on Dec 15, 2006
Title TSA + GW3965 + LPS
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 were normalized using a multi-loess technique described in Corbeil et al (Journal of Molecular Endocrinology (2004) 33:1–9).
 
Submission date Nov 22, 2006
Last update date Nov 24, 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)
GSE6352 The activation of inflammatory response genes by LPS is blocked by HDAC inhibitor Trichostatin A (TSA)

Data table header descriptions
ID_REF
VALUE Intensity

Data table
ID_REF VALUE
1 0.176316842
2 141.8894982
3 2.6353927
4 0.076750463
5 0.135252368
6 27.79340446
7 4.125954996
8 0.084463131
9 16.73109169
10 3.116625274
11 83.43807241
12 0.439358048
13 11.44180975
14 5.672722231
15 7.217267598
16 4.855287606
17 0.98786018
18 0.047344035
19 0.117121902
20 0.184574745

Total number of rows: 10076

Table truncated, full table size 165 Kbytes.




Supplementary data files not provided

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