NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM288378 Query DataSets for GSM288378
Status Public on Aug 31, 2008
Title VectorTransfected-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) -0.028 0.980779004 0.980779004 11900 35260 120 357 367 138 37 90 90 95 40 41 99 93 0 449 451 112 24 176 176 178 41 23 100 94 0 0.981 1.009 1.018 0.973 1.568 1.046 0.704 120 795 100 539 551 267 272 277 274 44111 54172 6.756 6.512 0 0 0
10_ORF11 -0.144 0.905006463 0.905006463 12850 30770 120 5686 6324 3524 55 94 94 98 40 40 100 100 0 6351 6398 3406 53 173 173 175 33 19 100 100 0 0.905 1.001 0.997 1.012 1.578 1.013 0.933 120 792 100 11768 12453 5591 6177 6229 6224 758912 767764 155.049 184.667 0 0 0
100_PAN-AS 0.114 1.082224645 1.082224645 16650 30720 150 18212 16782 5127 30 109 109 116 53 46 100 100 0 16926 15877 4479 28 195 195 201 39 19 100 100 0 1.082 1.063 1.059 1.044 1.52 1.076 0.938 156 1034 100 34833 32354 18102 16731 16672 15682 2617957 2476884 309.4 393.667 0 0 0
101_UPPAN-A -0.031 0.97874165 0.97874165 16910 35200 120 1077 1135 500 44 111 111 116 46 39 100 100 0 1206 1177 483 41 220 220 223 37 16 97 96 0 0.979 1.07 1.086 1.192 1.972 1.065 0.873 120 772 100 1951 1979 965 986 1023 956 136170 141258 22.128 25.243 0 0 0
102_UPPAN-B 0.101 1.072516617 1.072516617 16910 30720 140 16609 16026 3645 22 110 110 117 47 40 100 100 0 15580 15224 3177 20 201 201 203 37 18 100 100 0 1.073 1.059 1.053 1.044 1.26 1.07 0.954 156 946 100 31878 30938 16499 15379 15915 15023 2500027 2374923 331.51 397.595 0 0 0
103_UPPAN-C -0.07 0.952637998 0.952637998 17150 35200 130 835 912 380 41 113 113 119 47 39 100 99 0 976 997 351 35 218 218 220 41 18 100 100 0 0.953 1.026 1.028 1.009 1.469 1.046 0.852 120 818 100 1478 1578 721 757 799 779 109571 119659 16.875 18.561 -100 0 0
104_UPPAN-D 0.092 1.065846736 1.065846736 17160 30720 150 7843 7083 2572 36 110 110 116 47 41 100 100 0 7461 7134 2190 30 204 204 206 36 17 100 100 0 1.066 1.006 1.003 0.939 1.514 1.034 0.929 156 984 100 14988 13902 7732 7256 6972 6930 1105045 1113029 145.184 188.472 0 0 0
105_UPPAN-E -0.034 0.976708529 0.976708529 17410 35200 140 2331 2256 618 27 110 110 115 46 39 99 99 0 2489 2400 659 27 215 215 217 41 19 100 100 0 0.977 0.982 0.972 0.978 1.707 0.976 0.916 156 942 100 4494 4330 2221 2273 2145 2185 352012 374477 46.511 52.146 0 0 0
106_UPPAN-F 0.101 1.072516617 1.072516617 17410 30720 140 12394 11572 3958 34 110 110 116 47 40 100 100 0 11660 11264 3649 32 206 206 209 38 18 100 100 0 1.072 1.036 1.04 1.017 1.399 1.048 0.918 156 916 100 23738 22519 12284 11454 11461 11058 1805311 1757214 243.688 284.921 0 0 0
11_K2 0.022 1.015366101 1.015366101 13160 35260 130 2026 1988 570 28 96 96 105 75 71 100 100 0 2081 2014 489 24 181 181 186 37 20 100 100 0 1.015 1.032 1.061 1.011 1.285 1.052 0.912 120 878 100 3829 3723 1929 1900 1891 1832 238543 241748 24.961 48.378 0 0 0
12_ORF2 -0.083 0.944092419 0.944092419 13110 30770 110 2275 2402 1397 58 94 94 104 79 75 100 100 0 2480 2549 1318 51 171 171 177 68 38 100 100 0 0.944 0.97 0.948 0.898 1.458 0.985 0.908 80 656 100 4488 4685 2180 2308 2307 2378 192194 203941 28.963 34.672 0 0 0
13_K3 -0.004 0.997231251 0.997231251 13410 35260 120 920 923 296 32 98 98 104 44 42 100 100 0 1005 1013 261 25 181 181 189 54 28 100 100 0 0.997 0.991 0.968 0.972 1.352 1.016 0.879 120 796 100 1645 1656 821 824 824 832 110824 121682 18.578 15.226 0 0 0
14_ORF70 -0.174 0.886381699 0.886381699 13350 30770 130 2260 2155 630 29 93 93 99 70 70 98 97 0 2619 2536 505 19 173 173 177 61 34 100 100 0 0.886 0.873 0.873 0.811 1.465 0.902 0.934 120 881 100 4612 4424 2167 2445 2062 2362 258710 304403 29.153 38.5 0 0 0
15_K4 0.048 1.033830736 1.033830736 13660 35260 120 742 770 220 28 94 94 98 39 39 100 100 0 803 803 189 23 176 176 178 31 17 100 100 0 1.034 1.079 1.104 1.075 1.298 1.093 0.865 120 802 100 1274 1302 648 626 676 626 92472 96447 17.15 19.742 100 0 0
16_K4_1 0.057 1.040300267 1.040300267 13600 30770 120 4191 4013 2436 60 93 93 99 41 41 99 99 0 4108 4093 2236 54 169 169 172 32 18 100 100 0 1.04 0.999 0.959 0.895 1.751 1.03 0.903 120 802 100 8037 7844 4098 3939 3920 3924 481575 491171 95.143 120 0 0 0
17_K4_2 -0.018 0.987600861 0.987600861 10910 35520 130 1289 1328 310 23 93 93 97 41 42 100 100 0 1379 1386 320 23 167 167 170 40 23 100 100 0 0.987 1.014 1.014 1.006 1.237 1.019 0.894 120 892 100 2408 2454 1196 1212 1235 1219 159513 166389 29.929 29.775 0 0 0
18_K5 0.022 1.015366101 1.015366101 10850 31030 130 7735 7424 4110 55 90 90 94 38 40 100 100 0 7694 7128 3404 47 166 166 166 30 18 100 100 0 1.016 1.054 1.064 0.917 1.704 1.1 0.927 120 896 100 15173 14295 7645 7528 7334 6961 890952 855383 191.897 227.267 0 0 0
19_K6 -0.024 0.983502074 0.983502074 11160 35520 130 1170 1117 456 40 94 94 97 42 43 100 99 0 1265 1198 413 34 170 170 171 34 20 100 99 0 0.983 0.995 1.014 0.968 1.44 1.019 0.902 120 886 100 2170 2051 1076 1094 1023 1028 134145 143883 24.209 29.588 0 0 0
2_K1(subgroupB) 0.215 1.160703914 1.160703914 11840 30770 120 840 805 292 36 95 95 104 69 66 100 98 0 808 813 245 30 167 167 173 53 30 98 96 0 1.161 1.097 1.087 1.095 1.542 1.129 0.852 120 799 100 1385 1355 744 641 709 646 96573 97598 10.07 12.058 -100 0 0
20_K7 0.126 1.091263877 1.091263877 11100 31020 120 761 1434 2223 154 91 91 97 40 41 99 97 0 780 1433 1978 138 165 165 167 32 19 99 99 0 1.091 1.06 1.17 1.033 1.914 1.124 0.7 120 802 100 1284 2611 670 614 1343 1268 172216 172015 33.293 38.75 -100 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

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap