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Sample GSM71435 Query DataSets for GSM71435
Status Public on Dec 01, 2005
Title HU 12 hrs KO - mAdbID:48457
Sample type RNA
 
Channel 1
Source name Untreated KO
Organism Homo sapiens
Characteristics cell_line: HCT116 p53-/-
cell_type: colon cancer
genetic_variation: gene knock out
Growth protocol protocol_name: Cell culture
media: McCoy's 5A medium containing 10% FBS, 4 mM glutamine, 10 u/ml penicillin, 10 ug/ml streptomycin
Extracted molecule total RNA
Extraction protocol protocol_name: RNA extraction with TRIZOL
extraction_method: TRIZOL
other: Cells were lysed with TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated according to the manufacturer’s instructions. RNA integrity was assessed by the presence of 28S and 18S bands in denaturing agarose gel eletrophoresis and spectrophotometry with A260/A280 ratios > 2.0 after dilution in TE-buffer.
Label cy3
Label protocol protocol_name: Cy3 Label Protocol
label_method: indirect labeling
other: For the preparation of fluorescence-labeled cDNA targets, 20 µg total RNA for each channel (Cy3 and Cy5) were reverse-transcribed using Superscript II enzyme reaction kit (Invitrogen, Carlsbad, CA). Briefly, total RNA was mixed with 4 µg of oligo dT (20-mer) primer for a total volume of 25 µl, denatured at 70°C, and cooled to room temperature. Then, a mix containing 2.5 µl of 20 mM dNTP pool [10 mM of dATP, dGTP, dCTP, and 4 mM of dTTP (Amersham), and 6 mM of aminoallyl-dUTP (Sigma-Aldrich, St. Louis, MO)], 10 µl of 5X first strand buffer (Invitrogen), 5 µl of 0.1M dithiothreitol (Invitrogen), 1.0 µl of Superase Inhibitor (Ambion, Austin, TX), 3.2 µl of dH2O, and 3.3 µl of Superscript II reverse transcriptase were added and incubated for 60 minutes at 42°C, followed by a 5-minute incubation at 75°C. Remaining RNA was degraded by adding 2 µl of RNAse H (2 U/µl) (Invitrogen), followed by incubation at 37°C for 20 minutes. To remove traces of protein, 10 µl of Quick Clean Enzyme Removal Resin (Clontech, Palo Alto, CA) were added to the cDNA, mixed by 1-minute vortex, and centrifuged at 14,000 rpm for 1 minute. The resulting supernatant was filtered through a 0.65 µm Ultra-free MC filter (Millipore, Bedford, MA) to remove any resin residues. The cDNA probes were precipitated by adding 5.5 µl of 3M sodium acetate (pH = 5.2) and 137.5 µl of cold 190-proof ethanol, and incubated for 60 minutes at -20°C, followed by centrifugation for 15 minutes at 14,000 rpm and 4°C. The cDNA precipitate was washed twice with 70% ethanol, air-dried, and dissolved with 17 µl of 0.1M sodium bicarbonate. For the dye-coupling reaction, the monoreactive Cy3 (for control probes) and Cy5 dyes (for treated probes) (Amersham) were dissolved with 2 µl of dH2O, mixed with the corresponding cDNA probe and incubated for 60 minutes in the dark. The labeled cDNA samples were again precipitated by adding 2.5 µl of 3 M sodium acetate (pH = 5.2) and 50 µl of cold 190-proof ethanol, followed by an incubation for 90 minutes, and a centrifugation for 10 minutes at 14,000 rpm and 4°C. The supernatant was discarded, probes were washed twice with 70% ethanol, and air-dried. To remove unincorporated dye residues, probes were cleaned-up using the Nucleospin Extraction Kit (Clontech) following the manufacturer’s recommendations. To further clean-up the probes, the eluate from the previous step was washed twice with TE buffer (tris ethylenediaminetetraacetic acid) (pH = 7.4) using a Microcon YM-30 column (Millipore).
 
Channel 2
Source name HU 12 hrs KO
Organism Homo sapiens
Characteristics cell_line: HCT116 p53-/-
cell_type: colon cancer
genetic_variation: gene knock out
Treatment protocol treatment_type: compound
compound: hydroxy urea
treatment_dose: 1.5 mM
treatment_time: 12 hrs
Growth protocol protocol_name: Cell culture
media: McCoy's 5A medium containing 10% FBS, 4 mM glutamine, 10 u/ml penicillin, 10 ug/ml streptomycin
Extracted molecule total RNA
Extraction protocol protocol_name: RNA extraction with TRIZOL
extraction_method: TRIZOL
other: Cells were lysed with TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated according to the manufacturer’s instructions. RNA integrity was assessed by the presence of 28S and 18S bands in denaturing agarose gel eletrophoresis and spectrophotometry with A260/A280 ratios > 2.0 after dilution in TE-buffer.
Label cy5
Label protocol protocol_name: Cy5 Label Protocol
label_method: indirect labeling
other: For the preparation of fluorescence-labeled cDNA targets, 20 µg total RNA for each channel (Cy3 and Cy5) were reverse-transcribed using Superscript II enzyme reaction kit (Invitrogen, Carlsbad, CA). Briefly, total RNA was mixed with 4 µg of oligo dT (20-mer) primer for a total volume of 25 µl, denatured at 70°C, and cooled to room temperature. Then, a mix containing 2.5 µl of 20 mM dNTP pool [10 mM of dATP, dGTP, dCTP, and 4 mM of dTTP (Amersham), and 6 mM of aminoallyl-dUTP (Sigma-Aldrich, St. Louis, MO)], 10 µl of 5X first strand buffer (Invitrogen), 5 µl of 0.1M dithiothreitol (Invitrogen), 1.0 µl of Superase Inhibitor (Ambion, Austin, TX), 3.2 µl of dH2O, and 3.3 µl of Superscript II reverse transcriptase were added and incubated for 60 minutes at 42°C, followed by a 5-minute incubation at 75°C. Remaining RNA was degraded by adding 2 µl of RNAse H (2 U/µl) (Invitrogen), followed by incubation at 37°C for 20 minutes. To remove traces of protein, 10 µl of Quick Clean Enzyme Removal Resin (Clontech, Palo Alto, CA) were added to the cDNA, mixed by 1-minute vortex, and centrifuged at 14,000 rpm for 1 minute. The resulting supernatant was filtered through a 0.65 µm Ultra-free MC filter (Millipore, Bedford, MA) to remove any resin residues. The cDNA probes were precipitated by adding 5.5 µl of 3M sodium acetate (pH = 5.2) and 137.5 µl of cold 190-proof ethanol, and incubated for 60 minutes at -20°C, followed by centrifugation for 15 minutes at 14,000 rpm and 4°C. The cDNA precipitate was washed twice with 70% ethanol, air-dried, and dissolved with 17 µl of 0.1M sodium bicarbonate. For the dye-coupling reaction, the monoreactive Cy3 (for control probes) and Cy5 dyes (for treated probes) (Amersham) were dissolved with 2 µl of dH2O, mixed with the corresponding cDNA probe and incubated for 60 minutes in the dark. The labeled cDNA samples were again precipitated by adding 2.5 µl of 3 M sodium acetate (pH = 5.2) and 50 µl of cold 190-proof ethanol, followed by an incubation for 90 minutes, and a centrifugation for 10 minutes at 14,000 rpm and 4°C. The supernatant was discarded, probes were washed twice with 70% ethanol, and air-dried. To remove unincorporated dye residues, probes were cleaned-up using the Nucleospin Extraction Kit (Clontech) following the manufacturer’s recommendations. To further clean-up the probes, the eluate from the previous step was washed twice with TE buffer (tris ethylenediaminetetraacetic acid) (pH = 7.4) using a Microcon YM-30 column (Millipore).
 
 
Hybridization protocol protocol_name: Oligo Microarray Hybridization
other: The probes were finally concentrated to a volume of 14 µl and mixed with 1 µl COT-1 DNA (8-10 µg/µl) (Invitrogen), 1 µl yeast tRNA (4 µg/µl) (Sigma), and 1 µl poly-dA DNA (8-10 µg/µl) (Amersham). Before hybridization, the probes were denatured for 2 minutes at 100?C and cooled to room temperature. We used 70-mer oligo glass slides featuring 22,149 spots, corresponding to approximately 9,800 unique Unigene Clusters (Qiagen Human Version 2.0 set), which were printed by the National Cancer Institute Microarray Facility, Advanced Technology Center, Gaithersburg, MD. Arrays were pre-hybridized for 60 minutes at 42°C using a pre-hybridization buffer consisting of 5X SSC (sodium chloride-sodium citrate), 1% bovine serum albumin, and 0.1% SDS (sodium dodecyl sulfate). They were then washed in dH2O, immersed in isopropanol for 2 minutes, and air-dried. Denatured probes were mixed at a 1:1 ratio with hybridization buffer consisting of 50% formamide (Ambion), 1X SSC, and 0.1% SDS, and hybridized onto the microarrays at 42°C for 14–16 hours in a water bath. After hybridization, the slides were washed for 1 minute in 1X SSC with 0.1% SDS, for 1 minute in 1X SSC, and twice for 1 minute in 0.2X SSC. The arrays were finally scanned at a 10µm resolution with a GenePix 4000A scanner (Axon Instruments, Foster City, CA) with variable voltages to achieve saturated signal intensities for about 1% of the total spot number.
Description mAdb experiment ID: 48457
Data processing After background correction and removal of flagged values, log base 2 expression ratios were median centered and linear transformed to obtain the log and linear values given in the data table.
 
Submission date Aug 22, 2005
Last update date Aug 23, 2006
Contact name Ana I Robles
E-mail(s) Ana_Robles@nih.gov
Phone 301-496-1729
Organization name National Cancer Institute/NIH
Lab Laboratory of Human Carcinogenesis
Street address 37 Convent Dr 37/3060D
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
 
Platform ID GPL1528
Series (1)
GSE3176 p53 In Inflamatory Stress Response

Data table header descriptions
ID_REF NCI mAdb well id plus replicate number
VALUE -[INV_VALUE]
PRE_VALUE Calibrated Ratio (Sample_target_source1 / Sample_target_source2 )
Slide_block Array block location
Slide_column Array column location
Slide_row Array row location
CH1_MEAN Sample 1 (Red Channel) mean Intensity
CH1_SD Sample 1 (Red Channel) Standard Deviation
CH1_BKD_median Sample 1 (Red Channel) median Background Level
CH1_BKD_SD Sample 1 (Red Channel) Background Standard Deviation
CH2_MEAN Sample 2 (Green Channel) mean Intensity
CH2_SD Sample 2 (Green Channel) Standard Deviation
CH2_BKD_median Sample 2 (Green Channel) median Background Level
CH2_BKD_SD Sample 2 (Green Channel) Background Standard Deviation
Flag Quality flag 0->good, -50->Not found, -100->Bad
UNF_VALUE log ratio (log2 of PRE_VALUE)
INV_VALUE same as UNF_VALUE but with flagged values removed

Data table
ID_REF VALUE PRE_VALUE Slide_block Slide_column Slide_row CH1_MEAN CH1_SD CH1_BKD_median CH1_BKD_SD CH2_MEAN CH2_SD CH2_BKD_median CH2_BKD_SD Flag UNF_VALUE INV_VALUE
1173588_1 0.211 0.864 1 1 1 372 86 117 35 382 94 105 44 0 -0.211 -0.211
1174364_1 0.002 0.999 1 1 2 7980 1702 117 30 7496 1222 108 52 0 -0.002 -0.002
1175152_1 0.04 0.973 1 1 3 398 99 116 32 379 103 107 62 0 -0.040 -0.040
1175940_1 -0.193 1.143 1 1 4 2867 499 117 32 2361 445 104 32 0 0.193 0.193
1176716_1 0.477 0.718 1 1 5 3820 720 119 33 4939 977 105 42 0 -0.477 -0.477
1177504_1 0.122 0.919 1 1 6 6571 1645 119 36 6695 1694 104 40 0 -0.122 -0.122
1178580_1 -0.176 1.130 1 1 7 4795 1162 122 34 3987 900 106 38 0 0.176 0.176
1179356_1 -0.097 1.070 1 1 8 230 57 124 33 204 56 111 49 0 0.097 0.097
1180144_1 0.028 0.981 1 1 9 6450 1630 124 37 6161 1364 109 46 0 -0.028 -0.028
1180932_1 -0.064 1.046 1 1 10 279 95 123 37 251 76 111 29 0 0.064 0.064
1181708_1 0.004 0.997 1 1 11 1394 408 127 35 1306 415 114 48 0 -0.004 -0.004
1182496_1 0.05 0.966 1 1 12 229 53 123 30 215 46 112 34 0 -0.050 -0.050
1183572_1 -0.204 1.152 1 1 13 4213 1469 120 30 3450 1100 115 32 0 0.204 0.204
1184348_1 -0.153 1.112 1 1 14 3515 869 120 31 2979 605 114 37 0 0.153 0.153
1185136_1 0.701 1 1 15 180 144 121 29 191 189 112 36 -50 -0.513
1185924_1 -0.368 1.290 1 1 16 394 85 119 33 311 82 111 39 0 0.368 0.368
1186700_1 -0.164 1.120 1 1 17 3217 724 121 33 2701 558 107 27 0 0.164 0.164
1187488_1 0.101 0.932 1 1 18 2601 746 124 33 2603 771 110 41 0 -0.101 -0.101
1188564_1 0.052 0.964 1 1 19 14870 5257 124 31 14455 4625 106 28 0 -0.052 -0.052
1189340_1 -0.081 1.058 1 1 20 1164 295 121 34 1029 242 104 30 0 0.081 0.081

Total number of rows: 21794

Table truncated, full table size 1593 Kbytes.




Supplementary data files not provided

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