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Sample GSM288361 Query DataSets for GSM288361
Status Public on Aug 31, 2008
Title RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Plus/Minus4-OHT-Sample3-Repl-1
Sample type RNA
 
Channel 1
Source name RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Plus 4-OHT-Sample3-Repl-1
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 RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Minus 4-OHT-Sample3-Repl-1
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 VALUE_Linear 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 the Y-coordinate in µm of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image.
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 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) -0.309 0.807201075 0.807201075 10970 33530 130 599 616 173 28 139 139 150 74 49 100 99 0 740 733 146 19 170 170 175 51 29 100 100 0 0.807 0.847 0.856 0.82 1.474 0.847 0.672 120 898 100 1030 1040 460 570 477 563 73919 87963 6.297 10.941 0 0 0
10_ORF11 0.052 1.036701101 1.036701101 11900 29030 140 2983 2803 957 34 150 150 159 73 45 96 94 0 2929 2656 925 34 196 196 202 56 27 94 93 0 1.037 1.078 1.073 1.098 1.36 1.06 0.933 156 997 100 5566 5113 2833 2733 2653 2460 437300 414407 36.219 43.821 0 0 0
100_PAN-AS 0.441 1.35754498 1.35754498 15680 29000 130 1651 1663 277 16 144 144 153 76 49 100 100 0 1329 1312 199 15 219 219 225 62 27 100 100 0 1.358 1.39 1.371 1.391 1.281 1.439 0.881 120 883 100 2617 2612 1507 1110 1519 1093 199606 157498 19.868 17.532 0 0 0
101_UPPAN-A -0.191 0.875998315 0.875998315 15940 33510 140 867 885 232 26 138 138 150 75 50 99 98 0 1024 994 232 23 192 192 195 55 28 100 98 0 0.876 0.931 0.952 0.941 1.505 0.925 0.8 156 998 100 1561 1549 729 832 747 802 138110 155087 9.8 14.527 0 0 0
102_UPPAN-B -0.13 0.91383145 0.91383145 15930 29000 130 2329 2303 352 15 149 149 158 78 49 100 100 0 2601 2582 309 11 215 215 219 59 26 100 100 0 0.914 0.91 0.894 0.906 1.197 0.919 0.928 120 893 100 4566 4521 2180 2386 2154 2367 276302 309835 27.5 40.051 0 0 0
103_UPPAN-C -0.211 0.863938187 0.863938187 16190 33510 130 779 782 175 22 143 143 153 76 49 100 100 0 926 910 158 17 190 190 196 56 28 100 99 0 0.864 0.887 0.885 0.88 1.359 0.875 0.785 120 862 100 1372 1359 636 736 639 720 93813 109259 8.276 12.75 0 0 0
104_UPPAN-D -0.133 0.911933166 0.911933166 16180 29000 130 3349 3326 509 15 150 150 162 80 49 100 100 0 3721 3643 499 13 212 212 217 58 26 100 100 0 0.912 0.926 0.935 0.924 1.165 0.929 0.932 120 892 100 6708 6607 3199 3509 3176 3431 399140 437192 39.55 59.069 0 0 0
105_UPPAN-E -0.103 0.93109482 0.93109482 16440 33510 140 2808 2684 802 29 147 147 154 72 46 98 97 0 3051 2826 793 28 194 194 200 58 29 100 99 0 0.931 0.964 0.943 0.952 1.617 0.968 0.907 156 1038 100 5518 5169 2661 2857 2537 2632 418734 440785 35.139 45.276 0 0 0
106_UPPAN-F 0.171 1.125838586 1.125838586 16430 29000 130 973 960 217 22 150 150 159 78 49 100 100 0 940 946 156 16 209 209 217 69 31 100 100 0 1.126 1.099 1.081 1.086 1.403 1.152 0.772 120 882 100 1554 1547 823 731 810 737 115213 113517 10.269 10.565 0 0 0
11_K2 0.154 1.112650121 1.112650121 12220 33530 140 1532 1498 469 31 141 141 152 78 51 99 98 0 1427 1365 424 31 177 177 181 54 29 98 98 0 1.113 1.142 1.14 1.176 1.714 1.141 0.863 156 1038 100 2641 2545 1391 1250 1357 1188 233696 212976 17.256 21.926 0 0 0
12_ORF2 -0.077 0.948026965 0.948026965 12160 29040 140 699 728 251 34 150 150 159 74 46 99 95 0 778 799 208 26 199 199 204 57 27 100 99 0 0.948 0.963 0.972 0.909 1.738 1.016 0.76 156 1012 100 1128 1178 549 579 578 600 113644 124630 7.689 10.439 0 0 0
13_K3 -0.15 0.901250463 0.901250463 12470 33540 130 1162 1158 233 20 147 147 157 74 47 100 100 0 1303 1299 183 14 177 177 182 53 29 100 100 0 0.901 0.901 0.923 0.888 1.25 0.916 0.875 120 850 100 2141 2133 1015 1126 1011 1122 138946 155834 13.527 21.075 0 0 0
14_ORF70 -0.065 0.955945318 0.955945318 12410 29030 130 1921 1955 413 21 146 146 156 74 47 100 100 0 2051 2047 342 16 194 194 202 74 36 100 100 0 0.956 0.976 0.981 0.97 1.288 0.984 0.892 120 898 100 3632 3662 1775 1857 1809 1853 234548 245611 24.311 24.932 0 0 0
15_K4 -0.16 0.895025071 0.895025071 12710 33530 140 666 652 239 36 146 146 155 72 46 94 91 0 755 729 218 29 174 174 179 54 30 96 93 0 0.895 0.912 0.906 0.891 2.036 0.949 0.704 156 1024 100 1101 1061 520 581 506 555 101649 113686 6.903 10.185 0 0 0
16_K4_1 0.014 1.009751298 1.009751298 12660 29030 130 1065 1072 240 22 146 146 155 75 48 100 100 0 1102 1104 216 19 192 192 196 57 29 100 99 0 1.01 1.015 1.049 1.015 1.441 1.029 0.843 120 872 100 1829 1838 919 910 926 912 128631 132426 12.227 15.93 0 0 0
17_K4_2 -0.144 0.905006463 0.905006463 12960 33530 130 1684 1726 296 17 142 142 155 75 48 100 100 0 1878 1860 333 17 174 174 177 53 29 100 100 0 0.905 0.94 0.921 0.948 1.336 0.924 0.901 120 892 100 3246 3270 1542 1704 1584 1686 207063 223194 20.947 31.755 0 0 0
18_K5 0.28 1.214194884 1.214194884 12910 29030 140 1212 1126 399 35 140 140 150 74 49 95 93 0 1080 1008 312 30 197 197 200 57 28 95 93 0 1.214 1.216 1.196 1.232 1.473 1.228 0.863 156 1012 100 1955 1797 1072 883 986 811 175586 157187 13.189 14.175 0 0 0
19_K6 -0.15 0.901250463 0.901250463 13210 33530 130 1038 1068 264 24 137 137 150 72 48 100 100 0 1177 1162 234 20 177 177 181 55 30 100 100 0 0.901 0.945 0.913 0.942 1.47 0.947 0.818 120 893 100 1901 1916 901 1000 931 985 128141 139385 12.75 17.836 0 0 0
2_K1(subgroupB) -0.093 0.937571096 0.937571096 10910 29040 140 1615 1571 389 24 146 146 159 78 49 100 100 0 1762 1683 400 23 195 195 202 56 27 100 99 0 0.937 0.958 0.939 0.967 1.429 0.957 0.899 156 1020 100 3036 2913 1469 1567 1425 1488 245088 262481 18.103 26.446 0 0 0
20_K7 0.205 1.152686347 1.152686347 13160 29040 140 1086 1095 313 28 149 149 160 79 49 99 98 0 1012 990 313 31 199 199 202 56 27 98 96 0 1.153 1.196 1.15 1.268 1.767 1.176 0.825 156 998 100 1750 1737 937 813 946 791 170830 154491 11.835 14.071 0 0 0

Total number of rows: 107

Table truncated, full table size 26 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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