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Sample GSM288340 Query DataSets for GSM288340
Status Public on Aug 31, 2008
Title VectorTransfected-Vero-rKSHV.219cells-Hygro-Plus/Minus4-OHT-Sample2A-Repl-2
Sample type RNA
 
Channel 1
Source name Vector-Transfected-Vero-rKSHV.219cells-Hygro-Plus 4-OHT-Sample2A-Repl-2
Organisms Chlorocebus aethiops; Human gammaherpesvirus 8
Characteristics Vero-rKSHV.219 cells are Vero cells infected with recombinant Human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus.
Biomaterial provider Jeffrey Vieira, Ph.D. Seattle, WA, USA
Treatment protocol For transfection, cells were seeded at 1x10^6 per 10-cm plate to obtain ~50 to 70% confluence the following day, and transfected with empty ER vector using TransIT-LT1 transfection reagent with 10 ug DNA according to the manufacturer’s (Mirus) instructions. Hygromycin B (300 ug/mL; Invitrogen) was added to cultures 3 hours post-transfection, and 4-hydroxytamoxifen (4-OHT; 0.1 mM; Sigma), was added 15 hours post-hygromycin. Total RNA was harvested 15 hours following 4-OHT addition.
Growth protocol Vero-rKSHV.219 cells were grown in complete Dulbecco’s modified Eagle medium (DMEM) supplemented with 6ug/ml puromycin as described in Vieira and O'Hearn (Virology 325; 225-240).
Extracted molecule total RNA
Extraction protocol Extraction and purification by RNABee (Tel-test), as recommended by manufacturer.
Label cyanine 5-dUTP [Cy5]
Label protocol 3 or 6 ugs. of each RNA sample (equal amounts per pair) were adjusted to 11 uL with dH2O. 2.2 ul of random hexamers (2 ug/ul; Sigma-Genosys) were added, the mixture was heated at 98°C for 2 minutes, then snapped cool on ice. 11.1 uL of master mix (5.0 uL First-strand buffer, 2.5 uL 100mM DTT, 2.3 uL low dTdNTP mix [5mM A,G,C and 0.2mM T stock], containing 1.5 uL cyanine 5-dUTP [Cy5] [PerkinElmer]) was added. The mixture was subsequently supplemented with 1.2 uL Superscript (Invitrogen), then incubated for 10 minutes at 25°C, followed by 90 min at 42°C.
 
Channel 2
Source name Vector-Transfected-Vero-rKSHV.219cells-Hygro-Minus 4-OHT-Sample2A-Repl-2
Organisms Chlorocebus aethiops; Human gammaherpesvirus 8
Characteristics Vero-rKSHV.219 cells are Vero cells infected with recombinant Human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus.
Biomaterial provider Jeffrey Vieira, Ph.D. Seattle, WA, USA
Treatment protocol For transfection, cells were seeded at 1x10^6 per 10-cm plate to obtain ~50 to 70% confluence the following day, and transfected with empty ER vector using TransIT-LT1 transfection reagent with 10 ug DNA according to the manufacturer’s (Mirus) instructions. Hygromycin B (300 ug/mL; Invitrogen) was added to cultures 3 hours post-transfection. Total RNA was harvested 30 hours following hygromycin addition.
Growth protocol Vero-rKSHV.219 cells were grown in complete Dulbecco’s modified Eagle medium (DMEM) supplemented with 6ug/ml puromycin as described in Vieira and O'Hearn (Virology 325; 225-240).
Extracted molecule total RNA
Extraction protocol Extraction and purification by RNABee (Tel-test), as recommended by manufacturer.
Label cyanine 3-dUTP [Cy3]
Label protocol 3 or 6 ugs. of each RNA sample (equal amounts per pair) were adjusted to 11 uL with dH2O. 2.2 ul of random hexamers (2 ug/ul; Sigma-Genosys) were added, the mixture was heated at 98°C for 2 minutes, then snapped cool on ice. 11.1 uL of master mix (5.0 uL First-strand buffer, 2.5 uL 100mM DTT, 2.3 uL low dTdNTP mix [5mM A,G,C and 0.2mM T stock], containing 1.5 uL cyanine 3-dUTP [Cy3] [PerkinElmer]) was added. The mixture was subsequently supplemented with 1.2 uL Superscript (Invitrogen), then incubated for 10 minutes at 25°C, followed by 90 min at 42°C.
 
 
Hybridization protocol Microcon 10 columns (YM 10; Millipore) were primed with 400 uL 1x TE buffer (Tris-EDTA pH 8.0) by centrifugation at RT for 10 min. Paired Cy3-labeled and Cy5-labeled cDNAs were combined, added to the primed column, and purified by centrifugation. When approximately 25 uL of liquid remained above the membrane, an extra 200 uL 1x TE buffer was added to wash the column. Centrifugation was continued until approximately 4.5 uL liquid remained above the membrane. The purified, labeled cDNAs were transferred to a new tube and combined with an equal volume of hybridization mixture (0.5 uL 10mg/ml tRNA (Sigma), 0.5 uL 10mg/ml salmon sperm DNA (Sigma), 1.0 uL 20x SSC, 2.5 uL formamide (Fisher), and 1.0 uL 1% SDS). The mixture was heated for 2 min at 98°C, and cooled for 5 min at RT immediately before loading on the microarray slides.
Prior to hybridization, the slides were blocked in pre-hybridization solution (3.0 g bovine serum albumin [fraction V; Roche], 1.2ml 10% SDS, 106.0 ml Milli-Q water) at 42°C for 1 h. The pre-hybridized slides were washed with nuclease-free water for 2 min, isopropanol for 2 min, and subsequently dried by centrifugation. The labeled cDNAs were applied to slides, covered by a 22 x 22-mm cover-slip (Corning), sealed in a hybridization chamber (GeneMachines), and hybridized at 50°C overnight. After hybridization, cover-slips were removed by submerging the slides in solution I (2x SSC, 1% SDS), rinsed in solution II (1x SSC, 0.05%SDS) for 1 min, and rinsed in solution III (0.06x SSC) for 2 min.
Scan protocol Slides were dried by centrifugation and scanned immediately with a GenePix4000B scanner (Molecular Devices). The images were processed using GenePix 5.1. Data were filtered by removing all spots that were below the background noise or flagged as ‘bad’. Spots were considered to be below the background noise if the sum of the median intensities of the two channels was less than twice the highest mean background of the chip. The chips were normalized by the print-tip Lowess method.
Description none
Data processing The ratio of the mean median intensity of Cy5 over the mean median intensity of Cy3 was determined for each spot.
Dye ratios for viral genes from the same transfections that were hybridized to greater than one independent micro-array were internally normalized to the averaged dye ratios of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), then viral spots were averaged. Each viral dye ratio was normalized to that of the KSHV true late ORF25/Major capsid protein transcript (as quantitated by Northern blotting, the MCP transcript was unaffected by Rta-ER WT and mutants in presence of protein synthesis inhibitors).
The five resulting data sets were tested for significant changes in gene expression using two methods: Method 1. Average number of standard deviations from mean of each data set: dye ratios of each spot for Rta_NLS1,2-ER were divided by dye ratios for ER vector to determine spot-specific effects of 4-OHT addition. Means of the triplicate dye ratios were calculated to determine gene specific effects of 4-OHT addition. Each data set was log transformed and fit to a normal (Gaussian) distribution, and the means of the normalized gene-specific values were calculated. The resulting set of gene-specific means was fit to a normal distribution, outliers were excluded using Peirce’s criterion (hypothesis: one outlier per gene), and the average number of standard deviations from the mean was calculated for each gene. Method 2. q-values: gene specific t tests and q values (chance of being false positive, expressed as a percentage) were calculated by comparing the dye ratios of each spot for Rta_NLS1,2-ER to the dye ratios for ER vector using the software program significance analysis of microarrays (SAM).
 
Submission date May 13, 2008
Last update date Aug 31, 2008
Contact name David M. Lukac
E-mail(s) lukacdm@njms.rutgers.edu
Phone 973 972-4868
Organization name Rutgers Univ./NJ Medical School
Department Microbiology, Biochemistry and Molecular Genetics
Street address 225 Warren St.; ICPH E350C; PO Box 1709
City Newark
State/province NJ
ZIP/Postal code 07101
Country USA
 
Platform ID GPL6852
Series (1)
GSE11432 Direct transcriptional targets of the HHV 8/KSHV Rta Lytic Switch Protein

Data table header descriptions
ID_REF
VALUE_median log (base 2) transform of the ratio of the medians.
VALUE_Linear inverse log of VALUE
VALUE_GAPDHNorm VALUE_Linear normalized to average of 3 replicates of VALUE_Linear of GAPDH
VALUE VALUE_GAPDHNorm normalized to average of 3 replicates of VALUE_Linear of ORF25
X the X-coordinate in µm of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image.
Y t
Dia. the diameter in µm of the feature-indicator.
F635 Median median feature pixel intensity at wavelength #1 (635 nm).
F635 Mean mean feature pixel intensity at wavelength #1 (635 nm).
F635 SD the standard deviation of the feature pixel intensity at wavelength #1 (635 nm).
F635 CV the coefficient of variation of the feature pixel intensity at wavelength #1 (635 nm).
B635 total background pixel intensity at wavelength #1 (635 nm).
B635 Median the median feature background intensity at wavelength #1 (635 nm).
B635 Mean the mean feature background intensity at wavelength #1 (635 nm).
B635 SD the standard deviation of the feature background intensity at wavelength #1 (635 nm).
B635 CV the coefficient of variation of the background pixel intensity at wavelength #1 (635 nm).
% > B635+1SD the percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength #1 (635 nm).
% > B635+2SD the percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength #1 (635 nm).
F635 % Sat. the percentage of feature pixels at wavelength #1 that are saturated.
F532 Median median feature pixel intensity at wavelength #2 (532 nm).
F532 Mean mean feature pixel intensity at wavelength #2 (532 nm).
F532 SD the standard deviation of the feature intensity at wavelength #2 (532 nm).
F532 CV the coefficient of variation of the feature pixel intensity at wavelength #2 (532 nm).
B532 total background pixel intensity at wavelength #2 (532 nm).
B532 Median the median feature background intensity at wavelength #2 (532 nm).
B532 Mean the mean feature background intensity at wavelength #2 (532 nm).
B532 SD the standard deviation of the feature background intensity at wavelength #2 (532 nm).
B532 CV the coefficient of variation of the background pixel intensity at wavelength #2 (532 nm).
% > B532+1SD the percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength #2 (532 nm).
% > B532+2SD the percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength #2 (532 nm).
F532 % Sat. the percentage of feature pixels at wavelength #2 that are saturated.
Ratio of Medians (635/532) the ratio of the median intensities of each feature for each wavelength, with the median background subtracted.
Ratio of Means (635/532) the ratio of the arithmetic mean intensities of each feature for each wavelength, with the median background subtracted.
Median of Ratios (635/532) the median of pixel-by-pixel ratios of pixel intensities, with the median background subtracted.
Mean of Ratios (635/532) the geometric mean of the pixel-by-pixel ratios of pixel intensities, with the median background subtracted.
Ratios SD (635/532) the geometric standard deviation of the pixel intensity ratios.
Rgn Ratio (635/532) the regression ratio of every pixel in a 2-feature-diameter circle around the center of the feature.
Rgn R2 (635/532) the coefficient of determination for the current regression value.
F Pixels the total number of feature pixels.
B Pixels the total number of background pixels.
Circularity a measure of circularity from 0 to 100, using a metric based on the variance of the distance of each boundary pixel to the centroid of the feature: 100 is most circular, 0 is most non-circular.
Sum of Medians (635/532) the sum of the median intensities for each wavelength, with the median background subtracted.
Sum of Means (635/532) the sum of the arithmetic mean intensities for each wavelength, with the median background subtracted.
F635 Median - B635 the median feature pixel intensity at 635 nm. with the median background subtracted.
F532 Median - B532 the median feature pixel intensity at 532 nm. with the median background subtracted.
F635 Mean - B635 the mean feature pixel intensity at 635 nm. with the median background subtracted.
F532 Mean - B532 the mean feature pixel intensity at 532 nm. with the median background subtracted.
F635 Total Intensity the sum of all pixel intensities in the feature at 635 nm.
F532 Total Intensity the sum of all pixel intensities in the feature at 532 nm.
SNR 635 the signal-to-noise ratio of the feature, calculated as (F635 Mean - B635 Mean) / B635 SD.
SNR 532 the signal-to-noise ratio of the feature, calculated as (F635 Mean - B635 Mean) / B635 SD.
Flags the type of flag associated with a feature.
Normalize flag column describing if the feature was used to calculate the normalization factors (1 for used, 0 for not used).
Autoflag reports whether or not a feature has been flagged from the Flag Features dialog box. It applies to good and bad flags only.

Data table
ID_REF VALUE_median VALUE_Linear VALUE_GAPDHNorm VALUE X Y Dia. F635 Median F635 Mean F635 SD F635 CV B635 B635 Median B635 Mean B635 SD B635 CV % > B635+1SD % > B635+2SD F635 % Sat. F532 Median F532 Mean F532 SD F532 CV B532 B532 Median B532 Mean B532 SD B532 CV % > B532+1SD % > B532+2SD F532 % Sat. Ratio of Medians (635/532) Ratio of Means (635/532) Median of Ratios (635/532) Mean of Ratios (635/532) Ratios SD (635/532) Rgn Ratio (635/532) Rgn R2 (635/532) F Pixels B Pixels Circularity Sum of Medians (635/532) Sum of Means (635/532) F635 Median - B635 F532 Median - B532 F635 Mean - B635 F532 Mean - B532 F635 Total Intensity F532 Total Intensity SNR 635 SNR 532 Flags Normalize Autoflag
1_K1(conserved) -1.718 0.303969821 0.755016096 0.755016096 10990 34960 110 12972 12859 2475 19 11674 11674 11786 1457 12 47 16 0 13865 13591 1559 11 9595 9595 9644 1139 11 93 86 0 0.304 0.297 0.469 0.469 2.726 0.71 0.427 80 656 100 5568 5181 1298 4270 1185 3996 1028759 1087253 0.736 3.465 0 0 0
10_ORF11 -1.982 0.253138702 0.628759111 0.628759111 11990 30450 130 16494 16456 1412 8 13102 13102 13188 1355 10 91 73 0 23996 23821 2622 11 10599 10599 10647 1128 10 100 99 0 0.253 0.254 0.246 0.237 1.673 0.267 0.517 120 868 100 16789 16576 3392 13397 3354 13222 1974715 2858526 2.412 11.679 0 0 0
100_PAN-AS -3.965 0.064034801 0.15905298 0.15905298 15730 30440 90 15301 15326 1392 9 15078 15078 15017 1107 7 34 5 0 16324 16295 1557 9 12842 12842 13074 1528 11 90 55 0 0.064 0.072 0.296 0.277 2.088 0.214 0.126 52 420 100 3705 3701 223 3482 248 3453 796954 847338 0.279 2.108 0 0 0
101_UPPAN-A -3.201 0.108743419 0.270102576 0.270102576 16000 34930 120 13767 13808 966 6 13474 13474 13386 1588 11 8 0 0 14815 14636 1457 9 12121 12121 12009 1313 10 80 51 0 0.109 0.133 0.257 0.262 3.39 0.623 0.199 120 786 100 2987 2849 293 2694 334 2515 1656921 1756358 0.266 2.001 0 0 0
102_UPPAN-B -2.817 0.141905263 0.352471693 0.352471693 16010 30440 110 16099 16108 1064 6 15309 15309 15224 1106 7 42 5 0 18599 18543 1433 7 13032 13032 12961 1030 7 100 97 0 0.142 0.145 0.184 0.156 2.404 0.212 0.24 80 656 100 6357 6310 790 5567 799 5511 1288611 1483468 0.799 5.419 0 0 0
103_UPPAN-C 16250 34930 130 13088 13015 1071 8 13261 13261 13311 1317 9 5 0 0 15326 15280 1544 10 12184 12184 12160 1159 9 90 75 0 -0.055 -0.079 0.203 0.18 3.312 0.382 0.192 120 887 100 2969 2850 -173 3142 -246 3096 1561853 1833633 -0.225 2.692 0 0 0
104_UPPAN-D -6.789 0.009042892 0.022461208 0.022461208 16260 30440 120 15448 15478 1438 9 15345 15345 15165 1429 9 17 2 0 24502 23934 3177 13 13109 13109 12964 1263 9 98 97 0 0.009 0.012 0.078 0.074 2.995 0.142 0.123 120 791 100 11496 10958 103 11393 133 10825 1857348 2872091 0.219 8.686 0 0 0
105_UPPAN-E -3.171 0.111028349 0.275778006 0.275778006 16510 34930 130 14865 14815 1255 8 13325 13325 13338 1384 10 59 10 0 26186 26024 2335 8 12316 12316 12289 1244 10 100 100 0 0.111 0.109 0.112 0.098 2.322 0.134 0.311 120 888 100 15410 15198 1540 13870 1490 13708 1777759 3122820 1.067 11.041 0 0 0
106_UPPAN-F 16500 30420 120 15394 14987 2235 14 15967 15967 15812 1541 9 6 0 0 15281 14957 2296 15 13537 13537 13448 1465 10 55 24 0 -0.329 -0.69 0.661 0.543 4.907 0.967 0.262 120 791 100 1171 440 -573 1744 -980 1420 1798486 1794836 -0.535 1.03 0 0 0
11_K2 -1.349 0.392564059 0.975071086 0.975071086 12250 34960 120 14414 14147 1931 13 12197 12197 12167 1161 9 73 47 0 15407 14914 1878 12 9760 9760 9776 969 9 95 93 0 0.393 0.378 0.455 0.381 2.192 0.549 0.508 120 804 100 7864 7104 2217 5647 1950 5154 1697589 1789656 1.705 5.302 0 0 0
12_ORF2 -1.297 0.406971593 1.010857269 1.010857269 12230 30450 110 14448 14407 1559 10 12865 12865 13094 1450 11 55 18 0 14314 14451 1550 10 10423 10423 10480 1068 10 98 87 0 0.407 0.383 0.459 0.374 2.633 0.635 0.399 80 656 100 5474 5570 1583 3891 1542 4028 1152565 1156082 0.906 3.718 0 0 0
13_K3 -2.275 0.20661258 0.513195102 0.513195102 12500 34950 110 14952 14320 2009 14 13438 13438 13451 977 7 65 35 0 18196 17394 2408 13 10867 10867 10931 944 8 96 95 0 0.207 0.135 0.261 0.231 2.998 0.24 0.303 80 640 100 8843 7409 1514 7329 882 6527 1145568 1391525 0.889 6.846 0 0 0
14_ORF70 -2.423 0.186468003 0.463158953 0.463158953 12480 30450 120 15684 15448 1348 8 13558 13558 13621 1336 9 73 26 0 22276 21879 2380 10 10878 10878 10881 1051 9 99 99 0 0.187 0.172 0.193 0.164 2.688 0.224 0.446 120 808 100 13524 12891 2126 11398 1890 11001 1853707 2625536 1.368 10.464 0 0 0
15_K4 -1.475 0.359733395 0.89352457 0.89352457 12750 34960 120 15246 15160 968 6 13636 13636 13728 994 7 77 30 0 15889 15785 1071 6 11413 11413 11409 878 7 99 98 0 0.36 0.349 0.37 0.342 1.823 0.362 0.475 120 798 100 6086 5896 1610 4476 1524 4372 1819144 1894195 1.441 4.984 0 0 0
16_K4_1 -0.995 0.501735874 1.246237735 1.246237735 12730 30450 120 15711 15468 1158 7 13792 13792 13803 873 6 76 54 0 14954 14874 1263 8 11128 11128 11087 864 7 97 94 0 0.502 0.447 0.479 0.418 1.972 0.485 0.51 120 792 100 5745 5422 1919 3826 1676 3746 1856147 1784934 1.907 4.383 0 0 0
17_K4_2 -2.155 0.224533093 0.557707008 0.557707008 12990 34960 120 15568 15310 1548 10 13272 13272 13372 1030 7 79 55 0 21334 20863 2395 11 11110 11110 11128 992 8 99 98 0 0.225 0.209 0.227 0.203 1.886 0.225 0.456 120 792 100 12520 11791 2296 10224 2038 9753 1837182 2503513 1.882 9.814 0 0 0
18_K5 -1.672 0.313817998 0.779477511 0.779477511 12980 30450 100 15197 15142 942 6 13829 13829 13863 871 6 70 37 0 15401 15486 1381 8 11041 11041 11034 904 8 100 97 0 0.314 0.295 0.308 0.263 2.619 0.363 0.436 80 560 100 5728 5758 1368 4360 1313 4445 1211331 1238884 1.468 4.925 0 0 0
19_K6 -1.367 0.387696603 0.962981045 0.962981045 13240 34960 120 15069 14883 1440 9 13049 13049 13094 795 6 81 66 0 16174 15989 1509 9 10965 10965 10972 905 8 99 96 0 0.388 0.365 0.38 0.365 2.089 0.421 0.386 120 802 100 7229 6858 2020 5209 1834 5024 1785966 1918692 2.25 5.544 0 0 0
2_K1(subgroupB) -2.58 0.167240944 0.415401781 0.415401781 10990 30450 130 16689 16804 1662 9 13752 13752 13698 1034 7 93 74 0 28254 27675 2864 10 10695 10695 10672 934 8 100 100 0 0.167 0.18 0.183 0.169 1.579 0.201 0.655 120 798 100 20496 20032 2937 17559 3052 16980 2016511 3320973 3.004 18.204 0 0 0
20_K7 -2.161 0.223601225 0.555392387 0.555392387 13230 30450 140 15680 15721 1036 6 13684 13684 13635 949 6 87 55 0 19964 19468 2467 12 11040 11040 11017 1009 9 98 96 0 0.224 0.242 0.258 0.237 2.105 0.273 0.548 156 954 100 10920 10465 1996 8924 2037 8428 2452483 3037044 2.198 8.376 0 0 0

Total number of rows: 107

Table truncated, full table size 30 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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