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Sample GSM290648 Query DataSets for GSM290648
Status Public on Oct 16, 2009
Title LPS, biological rep3
Sample type RNA
 
Source name LPS
Organism Mus musculus
Characteristics cell line: Raw 264.7
Biomaterial provider ATCC
Treatment protocol Cells were stimulated with lipopolysaccharide (LPS) at a final concentration of 1 μg/mL
Growth protocol Cells were cultured under standard incubation conditions (37 ºC, 5% CO2) and grown in RPMI supplemented with 5% FBS and 1μg/mL P/S
Extracted molecule total RNA
Extraction protocol Cells were harvested at 24 h using Versagene RNA purification and DNase treatment kits. Six wells of a 6-well plate were pooled for each biological replicate.
Label Digoxigenin
Label protocol All RNA Preps were run on an Agilent 2100 Bioanalyzer to assess RNA integrity. Those samples meeting minimum requirements of RIN value of 7.0 and greater were used to generate targets for hybridization to ABI arrays. One (1) ug of Total RNA (30ng mRNA) was used to generate First Strand cDNA using the NanoAmp RT-IVT labeling kit according to manufacturer’s protocol (ABI, Cat#4365715). Following first strand synthesis, second strand synthesis was completed. The resulting cDNA was then purified using an ABI kit provided column and the entire reaction was used in an IVT reaction to generate DIG labeled cRNA. The cRNA was then purified using a kit provided column and assessed for quality on an Agilent Bioanalyzer. All reactions meeting ABI criteria in terms of quantity and size of target produced were fragmented and then hybridized to the appropriate array for 16 hours with agitation at 55C.
 
Hybridization protocol All hybridization reagents, hybridization controls, wash reagents, and chemiluminescent reagents were provided in the ABI CL Detection Kit, part#4342142, and the manufacturer’s protocol was followed. Briefly, the arrays were equilibrated to room temperature and then pre-hybridized with a 1ml volume for 60’ with agitation (100RPM) at 55C per manufacturer’s protocol. During the pre-hybridization, the DIG-labelled targets were fragmented and then stored on ice until the pre-hyb was completed. Once pre-hyb was completed the targets were mixed with the appropriate reagents, including Hybridization controls, and the 0.5ml target was added to the pre-hybridization through the port on the array chamber. The arrays were immediately returned to the 55C Hybridization oven and agitated exactly 16 hours at 100RPM. The arrays were then washed and incubated with Anti-Dig-APAntibody for 20 minutes. Following antibody washes, the arrays were incubated with Chemiluminescence Enhancing Solution, washed a final time and then stored in the last wash buffer at room temperature. Substrate for the chemiluminescence reaction was added to each array individually and then the array was immediately imaged on the 1700 Chemiluminescent Analyzer.
Scan protocol Following addition of the chemiluminescence reaction substrate, each array was immediately imaged on the 1700 Chemiluminescent Analyzer. Each imaging was completed in approximately 15 minutes and the images were assessed for QC/QA and a primary analysis was completed by the AB1700 Expression Array System Software (v 1.1.1).
Description DWW25
Data processing Signal intensities across microarrays were quantile normalized
 
Submission date May 22, 2008
Last update date Mar 03, 2009
Contact name David W Wright
E-mail(s) David.Wright@vanderbilt.edu
Phone 615-322-2636
Fax 615-343-1234
Organization name Vanderbilt University
Department Chemistry
Street address SC Stevenson Center 7, Station B 351822
City Nashville
State/province TN
ZIP/Postal code 37235-1822
Country USA
 
Platform ID GPL2995
Series (2)
GSE13281 Expression profiles of beta-hematin (BH)- or 4-hydroxy-2-nonenal (HNE)-treated RAW 264.7 cells
GSE15070 Expression Profiles of 15(S)-HETE treated RAW 264.7 cells

Data table header descriptions
ID_REF
VALUE Quantile normalized signal intensity but with flagged values removed
SIGNAL_RAW Raw signal intensity
SDEV Noise/background signal intensity
CV Coefficient of variation of signal intensity based on the error model built into the Applied Biosystems image analysis software
S/N Signal-to-Noise ratio. Used for probe detectability (a probe with S/N >= 3 is considered detected)
FLAG Quality of each probe (FLAG > 5000 indicates quantification error)
UNF_VALUE Quantile normalized signal intensity

Data table
ID_REF VALUE SIGNAL_RAW SDEV CV S/N FLAG UNF_VALUE
297784 107780.806 120112.31 1905.45 0.05 63.04 0 107780.806
297907 1204.142667 1397.49 393.77 0.28 3.55 0 1204.142667
297912 2857.652667 3531.76 364.15 0.11 9.7 0 2857.652667
297935 323.1126667 275.41 275.41 0.93 -1.08 1 323.1126667
297990 7467.380667 9140.59 325.24 0.06 28.1 0 7467.380667
297993 240.762 199.38 199.38 0.48 -2.08 1 240.762
298000 23162.23733 27047.47 635.72 0.04 42.55 0 23162.23733
298038 148.4393333 120.05 120.05 2.27 0.44 1 148.4393333
298121 422.424 377.73 377.73 1.65 0.61 1 422.424
298130 1464.004 1744.84 1744.84 1.69 0.59 1 1464.004
298143 524.0326667 502.48 207.53 0.42 2.42 0 524.0326667
298150 157.7626667 128.12 128.12 2.07 0.48 1 157.7626667
298151 412.5193333 367.1 367.1 1.15 0.87 1 412.5193333
298155 268.094 224.01 224.01 1.5 0.67 1 268.094
298165 628.3793333 637 276.85 0.44 2.3 0 628.3793333
298174 9672.849333 11799.48 381.19 0.05 30.95 0 9672.849333
298188 397.9333333 352.32 352.32 0.69 -1.45 1 397.9333333
298200 520342.6907 563706 15150.37 0.05 37.21 0 520342.6907
298246 318.886 271.6 271.6 0.66 -1.52 1 318.886
298248 356.2586667 308.56 308.56 0.56 -1.8 1 356.2586667

Total number of rows: 33012

Table truncated, full table size 1799 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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