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Sample GSM288383 Query DataSets for GSM288383
Status Public on Aug 31, 2008
Title RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Plus/Minus4-OHT-Sample5-Repl-3
Sample type RNA
 
Channel 1
Source name Vector-Transfected-Vero-rKSHV.219cells-Hygro-Plus 4-OHT-Sample5-Repl-3
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-Sample5-Repl-3
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) -0.029 0.980099415 0.995572374 11370 28640 120 2047 2288 1020 44 94 94 98 38 39 100 100 0 2069 2262 902 39 162 162 164 32 20 100 100 0 1.024 1.045 1.05 1.03 0.909 1.077 0.939 120 793 100 3860 4294 1953 1907 2194 2100 274702 271449 56.872 63.667 0 0 0
10_ORF11 0.091 1.065108203 1.081923207 12110 28640 110 2322 2288 1280 55 99 99 106 41 39 100 100 0 2364 2392 1172 49 173 173 176 36 20 100 100 0 1.014 0.986 0.963 0.925 1.074 1.053 0.853 80 656 100 4413 4407 2222 2191 2188 2219 183014 191456 52.196 60.533 -100 0 0
100_PAN-AS 0.128 1.092777739 1.110029566 12870 28630 130 6829 6596 3236 49 104 104 113 58 51 100 100 0 6634 6179 3183 51 174 174 179 57 31 100 100 0 1.041 1.081 1.095 1.151 0.96 1.076 0.939 120 894 100 13184 12496 6724 6460 6492 6004 791652 741533 111.437 104.617 0 0 0
101_UPPAN-A -0.122 0.918912883 0.933419882 13610 28620 140 2477 2629 1752 66 99 99 107 45 41 100 98 0 2549 2669 1624 60 178 178 180 40 22 100 100 0 1.003 1.015 1.034 0.963 1.105 1.056 0.916 156 1062 100 4747 5020 2377 2370 2529 2491 410183 416519 55.964 61.818 -100 0 0
102_UPPAN-B -0.019 0.986916546 1.002497128 11360 28880 120 1177 1322 619 46 90 90 95 37 39 100 99 0 1308 1338 621 46 161 161 163 32 20 100 98 0 0.947 1.046 1.055 1.078 0.969 1.048 0.924 120 787 100 2233 2408 1086 1147 1231 1177 158676 160562 32.565 35.667 0 0 0
103_UPPAN-C -0.069 0.953298545 0.968348395 12110 28880 120 3813 3516 1749 49 98 98 102 40 40 100 100 0 3665 3575 1749 48 168 168 170 34 20 100 100 0 1.062 1.003 1.007 1.001 1.02 1.015 0.93 120 802 100 7211 6824 3714 3497 3418 3406 421937 429093 83.32 99.643 0 0 0
104_UPPAN-D 0.059 1.041743429 1.05818957 12860 28880 120 2405 2718 2033 74 104 104 112 47 42 100 97 0 2612 2852 2026 71 180 180 186 64 34 99 98 0 0.946 0.978 0.97 0.945 1.226 1.008 0.896 120 796 100 4731 5285 2300 2431 2613 2672 326180 342306 54.828 41.226 -100 0 0
105_UPPAN-E -0.081 0.945402117 0.960327305 13610 28880 120 943 974 378 38 99 99 104 39 37 99 98 0 988 1009 372 36 173 173 175 35 20 100 100 0 1.034 1.046 1.075 1.016 1.114 1.06 0.892 120 802 100 1658 1709 843 815 874 835 116908 121083 22.104 23.552 0 0 0
106_UPPAN-F 0.074 1.052631155 1.069249182 11360 29130 120 3572 3309 989 29 95 95 99 36 36 100 100 0 3483 3287 866 26 161 161 163 32 20 100 100 0 1.046 1.028 1.017 1.001 0.898 1.051 0.956 120 808 100 6799 6339 3477 3322 3213 3126 397076 394512 89.023 94.815 0 0 0
11_K2 0.161 1.118061851 1.135712841 11390 29390 130 6950 6687 1628 24 94 94 99 38 38 100 100 0 6688 6135 1747 28 158 158 159 32 20 100 100 0 1.05 1.103 1.082 1.131 0.866 1.091 0.947 120 897 100 13385 12569 6856 6529 6593 5976 802498 736268 171.064 181.37 0 0 0
12_ORF2 -0.169 0.889458994 0.903501 13630 29890 130 4367 4414 1752 39 95 95 99 37 37 100 100 0 3990 4102 1629 39 158 158 162 34 21 100 100 0 1.115 1.095 1.083 1.104 0.929 1.1 0.954 120 892 100 8103 8261 4272 3831 4318 3943 529775 492286 114.457 115.321 0 0 0
13_K3 -0.083 0.944092419 0.958996931 12860 30640 100 3072 3092 1219 39 96 96 102 41 40 100 100 0 2957 3077 1155 37 164 164 168 42 25 100 100 0 1.065 1.029 1.013 1.018 0.834 1.05 0.964 80 560 100 5768 5908 2975 2793 2996 2912 247391 246198 71.549 68.114 0 0 0
14_ORF70 -0.09 0.939522749 0.954355119 12110 31390 120 1378 1380 363 26 92 92 99 45 45 100 100 0 1348 1361 357 26 163 163 168 39 23 100 100 0 1.085 1.075 1.078 1.081 0.856 1.088 0.937 120 798 100 2469 2486 1285 1184 1288 1198 165692 163381 27.911 30.563 0 0 0
15_K4 0.068 1.048262476 1.064811534 12870 31390 120 616 672 281 41 98 98 100 38 38 100 100 0 621 706 300 42 165 165 167 34 20 99 99 0 1.138 1.063 1.073 1.118 1.019 1.056 0.881 120 808 100 972 1114 517 455 574 540 80772 84830 14.851 15.786 0 0 0
16_K4_1 -0.042 0.971307496 0.986641655 16670 28840 140 4185 3972 1950 49 114 114 121 47 39 100 99 0 3854 3818 1664 43 213 213 215 41 19 100 100 0 1.118 1.07 1.058 0.999 0.994 1.109 0.944 156 901 100 7711 7461 4070 3641 3857 3604 619670 595723 81.017 86.824 0 0 0
17_K4_2 -0.029 0.980099415 0.995572374 15930 29580 140 7736 7385 2455 33 103 103 110 49 45 100 100 0 6915 6536 2297 35 176 176 181 42 23 100 100 0 1.133 1.145 1.144 1.182 0.987 1.153 0.935 156 998 100 14371 13642 7633 6738 7282 6360 1152101 1019779 145.525 148.8 0 0 0
18_K5 -0.015 0.989656656 1.005280497 18170 30080 140 1878 1914 550 28 176 176 186 77 41 100 100 0 2063 2078 546 26 252 252 255 47 18 100 100 0 0.94 0.952 0.966 0.939 0.85 0.981 0.922 156 984 100 3512 3563 1702 1810 1738 1825 298609 324159 22.189 38.308 0 0 0
19_K6 0.056 1.039579435 1.055991413 17410 30830 140 2029 2780 2262 81 105 105 114 59 51 97 96 0 2342 3004 2216 73 208 208 213 45 21 98 97 0 0.901 0.957 0.917 0.871 1.09 1 0.934 156 915 100 4057 5469 1923 2134 2674 2795 433769 468676 45.181 61.811 0 0 0
2_K1(subgroupB) 0.105 1.07549439 1.092473362 16640 31560 90 2284 2076 979 47 108 108 115 46 40 100 98 0 2359 2234 910 40 198 198 202 41 20 100 100 0 1.007 0.967 0.927 0.894 0.944 0.996 0.925 52 420 100 4337 4003 2176 2161 1968 2035 107986 116195 41.982 48.971 -100 0 0
20_K7 1.791 3.460546762 3.515178871 17410 31570 100 108 122 56 46 105 105 112 47 42 18 6 0 206 211 58 27 207 207 209 41 19 15 2 0 -2.686 4.477 0.883 0.93 2.924 2.501 0.083 80 560 100 2 19 3 -1 16 3 9743 16922 0.207 0.029 -50 0 0

Total number of rows: 107

Table truncated, full table size 27 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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