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Sample GSM288371 Query DataSets for GSM288371
Status Public on Aug 31, 2008
Title VectorTransfected-Vero-rKSHV.219cells-Hygro-Plus/Minus4-OHT-Sample4-Repl-3
Sample type RNA
 
Channel 1
Source name Vector-Transfected-Vero-rKSHV.219cells-Hygro-Plus 4-OHT-Sample4-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-Sample4-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) -0.43 0.742261785 0.742261785 12010 35400 130 433 442 154 34 188 188 200 101 50 85 60 0 619 622 149 23 289 289 295 78 26 98 90 0 0.742 0.763 0.825 0.701 2.456 0.808 0.367 120 903 100 575 587 245 330 254 333 53099 74640 2.396 4.192 0 0 0
10_ORF11 -0.042 0.971307496 0.971307496 12940 30910 150 1130 1550 1095 70 152 152 159 69 43 99 98 0 1246 1514 888 58 239 239 243 57 23 100 100 0 0.971 1.096 1.017 0.999 1.503 1.158 0.909 156 1032 100 1985 2673 978 1007 1398 1275 241831 236156 20.159 22.298 0 0 0
100_PAN-AS 0.235 1.176906737 1.176906737 16720 30870 140 1240 1226 280 22 167 167 177 77 43 100 100 0 1196 1196 228 19 284 284 292 80 27 100 99 0 1.177 1.161 1.143 1.158 1.418 1.182 0.834 156 975 100 1985 1971 1073 912 1059 912 191259 186571 13.623 11.3 0 0 0
101_UPPAN-A 0.019 1.0132569 1.0132569 16970 35380 130 682 686 162 23 160 160 188 357 189 83 14 0 793 800 151 18 278 278 389 1648 423 0 0 0 1.014 1.008 1.031 0.996 1.495 0.549 0.803 120 878 100 1037 1048 522 515 526 522 82268 96022 1.395 0.249 0 0 0
102_UPPAN-B -0.035 0.976031761 0.976031761 16970 30870 140 4799 4533 1353 29 166 166 178 79 44 100 99 0 5039 4882 1225 25 291 291 298 83 27 100 100 0 0.976 0.951 0.95 0.918 1.43 0.967 0.94 156 984 100 9381 8958 4633 4748 4367 4591 707204 761608 55.127 55.229 0 0 0
103_UPPAN-C -0.117 0.922103118 0.922103118 17220 35370 130 865 858 215 25 155 155 162 69 42 100 99 0 1036 1047 213 20 266 266 274 88 32 100 100 0 0.922 0.9 0.853 0.89 1.546 0.882 0.747 120 878 100 1480 1484 710 770 703 781 102975 125681 10.087 8.784 0 0 0
104_UPPAN-D 0.094 1.067325338 1.067325338 17220 30870 140 4138 4065 764 18 166 166 174 76 43 100 100 0 4006 3810 763 20 285 285 289 69 23 100 100 0 1.067 1.106 1.079 1.122 1.302 1.1 0.939 156 946 100 7693 7424 3972 3721 3899 3525 634097 594399 51.197 51.029 0 0 0
105_UPPAN-E 0.167 1.122721422 1.122721422 17470 35370 130 2613 2619 475 18 155 155 163 68 41 100 100 0 2469 2439 408 16 280 280 294 99 33 100 100 0 1.123 1.141 1.139 1.146 1.287 1.13 0.9 120 886 100 4647 4623 2458 2189 2464 2159 314332 292684 36.118 21.667 0 0 0
106_UPPAN-F 0.246 1.185914499 1.185914499 17470 30870 140 841 825 251 30 171 171 179 77 43 100 97 0 856 871 205 23 291 291 298 81 27 98 97 0 1.186 1.128 1.127 1.128 1.632 1.144 0.742 156 976 100 1235 1234 670 565 654 580 128648 135831 8.39 7.074 0 0 0
11_K2 0.232 1.174461971 1.174461971 13250 35400 130 1570 1532 396 25 184 184 196 83 42 99 98 0 1471 1433 355 24 291 291 301 85 28 99 97 0 1.175 1.18 1.18 1.194 1.445 1.153 0.835 120 844 100 2566 2490 1386 1180 1348 1142 183813 172014 16.096 13.318 0 0 0
12_ORF2 -0.296 0.814507563 0.814507563 13190 30910 140 698 709 211 29 154 154 165 73 44 99 98 0 920 899 183 20 252 252 254 59 23 100 100 0 0.814 0.858 0.829 0.823 1.524 0.876 0.781 156 946 100 1212 1202 544 668 555 647 110674 140303 7.452 10.932 0 0 0
13_K3 -0.138 0.908778116 0.908778116 13500 35400 120 1178 1165 298 25 170 170 183 78 42 100 100 0 1384 1367 294 21 275 275 282 70 24 100 100 0 0.909 0.911 0.918 0.894 1.467 0.925 0.821 120 783 100 2117 2087 1008 1109 995 1092 139765 164004 12.59 15.5 0 0 0
14_ORF70 -0.021 0.985549337 0.985549337 13440 30900 130 3217 3256 445 13 169 169 176 75 42 100 100 0 3353 3374 383 11 260 260 264 62 23 100 100 0 0.985 0.991 0.985 0.989 1.175 1.001 0.939 120 861 100 6141 6201 3048 3093 3087 3114 390757 404909 41.067 50.161 0 0 0
15_K4 -0.18 0.882702996 0.882702996 13760 35410 130 883 884 195 22 168 168 179 79 44 100 100 0 1078 1096 175 15 268 268 274 75 27 100 100 0 0.883 0.865 0.871 0.85 1.372 0.894 0.784 120 928 100 1525 1544 715 810 716 828 106024 131469 8.924 10.96 0 0 0
16_K4_1 -0.07 0.952637998 0.952637998 13690 30910 130 1044 1060 213 20 174 174 183 95 51 100 100 0 1177 1213 175 14 264 264 271 71 26 100 100 0 0.953 0.934 0.953 0.921 1.289 0.964 0.844 120 873 100 1783 1835 870 913 886 949 127185 145586 9.232 13.268 0 0 0
17_K4_2 -0.046 0.968618189 0.968618189 11000 35650 130 1960 1919 412 21 183 183 192 81 42 99 99 0 2115 2044 389 19 280 280 287 68 23 99 99 0 0.968 0.984 0.991 0.986 1.494 0.991 0.909 120 883 100 3612 3500 1777 1835 1736 1764 230313 245307 21.321 25.838 0 0 0
18_K5 -0.219 0.859160755 0.859160755 10930 31160 130 1414 1434 308 21 151 151 157 68 43 100 100 0 1713 1697 318 18 243 243 247 60 24 100 100 0 0.859 0.882 0.861 0.884 1.378 0.871 0.841 120 903 100 2733 2737 1263 1470 1283 1454 172138 203605 18.779 24.167 0 0 0
19_K6 -0.139 0.908148418 0.908148418 11250 35660 120 1049 1043 264 25 178 178 193 85 44 100 100 0 1244 1207 221 18 285 285 291 75 25 100 98 0 0.908 0.938 0.938 0.926 1.436 0.957 0.831 120 786 100 1830 1787 871 959 865 922 125205 144811 10 12.213 0 0 0
2_K1(subgroupB) 0.052 1.036701101 1.036701101 11940 30900 110 1201 1221 315 25 155 155 163 108 66 100 100 0 1247 1226 260 21 238 238 250 140 56 100 97 0 1.037 1.079 1.074 1.075 1.361 1.059 0.848 80 650 100 2055 2054 1046 1009 1066 988 97679 98105 9.796 6.971 0 0 0
20_K7 -0.106 0.929160674 0.929160674 11190 31160 130 740 761 172 22 148 148 157 72 45 100 100 0 880 887 134 15 243 243 249 59 23 100 100 0 0.929 0.952 0.968 0.935 1.436 0.966 0.742 120 876 100 1229 1257 592 637 613 644 91285 106447 8.389 10.814 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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