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Sample GSM288362 Query DataSets for GSM288362
Status Public on Aug 31, 2008
Title RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Plus/Minus4-OHT-Sample3-Repl-2
Sample type RNA
 
Channel 1
Source name RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Plus 4-OHT-Sample3-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 RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Minus 4-OHT-Sample3-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 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) -0.147 0.90312651 0.90312651 11480 34550 120 592 577 175 30 144 144 152 73 48 98 95 0 675 678 136 20 179 179 182 53 29 100 100 0 0.903 0.868 0.868 0.829 1.635 0.936 0.64 120 808 100 944 932 448 496 433 499 69253 81349 5.822 9.358 0 0 0
10_ORF11 0.143 1.104198847 1.104198847 12420 30060 130 2921 2948 449 15 149 149 157 76 48 100 100 0 2691 2700 294 10 181 181 185 55 29 100 100 0 1.104 1.111 1.089 1.104 1.216 1.123 0.925 120 867 100 5282 5318 2772 2510 2799 2519 353796 323956 36.724 45.727 0 0 0
100_PAN-AS 0.323 1.25092908 1.25092908 16200 30020 130 1298 1320 322 24 143 143 156 78 50 100 100 0 1110 1090 211 19 187 187 193 56 29 100 99 0 1.251 1.303 1.237 1.302 1.417 1.37 0.83 120 867 100 2078 2080 1155 923 1177 903 158415 130851 14.923 16.018 0 0 0
101_UPPAN-A -0.122 0.918912883 0.918912883 16450 34520 130 936 941 222 23 143 143 153 77 50 100 100 0 1063 1060 186 17 200 200 203 57 28 100 100 0 0.919 0.928 0.931 0.914 1.395 0.932 0.806 120 877 100 1656 1658 793 863 798 860 112936 127222 10.234 15.035 0 0 0
102_UPPAN-B -0.15 0.901250463 0.901250463 16450 30020 140 1971 1792 702 39 145 145 156 77 49 96 94 0 2218 1981 659 33 192 192 198 57 28 98 97 0 0.901 0.921 0.918 0.883 1.596 0.941 0.914 156 988 100 3852 3436 1826 2026 1647 1789 279498 308999 21.247 31.281 0 0 0
103_UPPAN-C -0.128 0.915099168 0.915099168 16700 34530 130 821 835 196 23 143 143 154 76 49 100 100 0 947 954 168 17 206 206 211 58 27 100 100 0 0.915 0.925 0.926 0.91 1.44 0.951 0.758 120 865 100 1419 1440 678 741 692 748 100155 114469 8.961 12.81 0 0 0
104_UPPAN-D -0.083 0.944092419 0.944092419 16700 30020 130 3358 3328 544 16 150 150 161 80 49 100 100 0 3596 3509 530 15 197 197 199 57 28 100 100 0 0.944 0.96 0.936 0.963 1.253 0.958 0.939 120 877 100 6607 6490 3208 3399 3178 3312 399409 421086 39.588 58.07 0 0 0
105_UPPAN-E -0.05 0.965936329 0.965936329 16950 34530 130 2855 2874 451 15 144 144 156 79 50 100 100 0 3008 3016 317 10 201 201 206 59 28 100 100 0 0.966 0.97 0.959 0.961 1.214 0.988 0.91 120 882 100 5518 5545 2711 2807 2730 2815 344883 361957 34.405 47.627 0 0 0
106_UPPAN-F 0.09 1.064370182 1.064370182 16950 30020 120 939 927 258 27 146 146 157 78 49 100 100 0 953 937 178 18 208 208 210 59 28 100 100 0 1.064 1.071 1.078 1.042 1.549 1.118 0.749 120 786 100 1538 1510 793 745 781 729 111233 112397 9.872 12.322 0 0 0
11_K2 0.246 1.185914499 1.185914499 12730 34540 130 1539 1504 381 25 143 143 155 76 49 100 100 0 1356 1302 318 24 179 179 184 54 29 98 97 0 1.186 1.212 1.205 1.252 1.489 1.196 0.85 120 898 100 2573 2484 1396 1177 1361 1123 180479 156274 17.75 20.704 0 0 0
12_ORF2 0.115 1.082975046 1.082975046 12670 30060 120 771 776 215 27 146 146 155 78 50 100 100 0 754 782 177 22 177 177 184 55 29 100 100 0 1.083 1.041 1.027 1.026 1.473 1.092 0.754 120 793 100 1202 1235 625 577 630 605 93108 93866 7.962 10.873 0 0 0
13_K3 -0.296 0.814507563 0.814507563 12990 34550 130 1054 1074 275 25 139 139 153 75 49 100 99 0 1300 1277 247 19 177 177 183 55 30 100 100 0 0.815 0.85 0.851 0.831 1.494 0.863 0.796 120 897 100 2038 2035 915 1123 935 1100 128911 153230 12.28 19.891 0 0 0
14_ORF70 -0.147 0.90312651 0.90312651 12930 30060 130 1887 1919 334 17 139 139 149 72 48 100 100 0 2114 2115 314 14 179 179 186 55 29 100 100 0 0.903 0.919 0.923 0.917 1.244 0.923 0.903 120 879 100 3683 3716 1748 1935 1780 1936 230257 253759 24.583 35.073 0 0 0
15_K4 -0.026 0.982139595 0.982139595 13240 34540 130 694 713 218 30 148 148 157 81 51 100 99 0 734 719 177 24 178 178 187 71 37 99 96 0 0.982 1.044 1.019 1.029 1.632 1.072 0.66 120 878 100 1102 1106 546 556 565 541 85608 86329 6.864 7.493 0 0 0
16_K4_1 -0.011 0.992404375 0.992404375 13170 30060 130 928 932 258 27 139 139 149 73 48 100 99 0 981 994 219 22 186 186 190 58 30 100 100 0 0.992 0.981 0.973 0.955 1.448 1.015 0.814 120 857 100 1584 1601 789 795 793 808 111799 119267 10.726 13.862 0 0 0
17_K4_2 -0.239 0.847332435 0.847332435 13480 34540 120 1625 1657 326 19 140 140 151 82 54 100 100 0 1938 1912 321 16 185 185 191 72 37 100 100 0 0.847 0.878 0.883 0.874 1.242 0.879 0.898 120 792 100 3238 3244 1485 1753 1517 1727 198797 229495 18.366 23.903 0 0 0
18_K5 0.04 1.028113827 1.028113827 13420 30050 130 960 977 252 25 150 150 158 77 48 100 100 0 977 966 187 19 189 189 194 59 30 100 100 0 1.028 1.064 1.061 1.049 1.425 1.102 0.808 120 887 100 1598 1604 810 788 827 777 117195 115866 10.636 13.085 0 0 0
19_K6 -0.233 0.85086373 0.85086373 13740 34550 140 909 911 360 39 145 145 157 78 49 96 94 0 1087 1027 343 33 189 189 192 56 29 96 94 0 0.851 0.914 0.832 0.89 2.141 0.941 0.807 156 1062 100 1662 1604 764 898 766 838 142059 160248 9.667 14.911 0 0 0
2_K1(subgroupB) 0.02 1.01395948 1.01395948 11420 30060 140 1549 1419 531 37 140 140 152 74 48 95 92 0 1572 1487 439 29 182 182 187 57 30 99 98 0 1.014 0.98 0.968 0.876 1.941 1.015 0.867 156 1010 100 2799 2584 1409 1390 1279 1305 221398 232049 17.122 22.807 0 0 0
20_K7 0.267 1.203303026 1.203303026 13680 30060 130 1153 1133 252 22 148 148 156 73 46 100 100 0 1015 1004 199 19 180 180 184 54 29 100 100 0 1.204 1.195 1.225 1.193 1.435 1.247 0.804 120 903 100 1840 1809 1005 835 985 824 135976 120456 13.384 15.185 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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