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Sample GSM288247 Query DataSets for GSM288247
Status Public on Aug 31, 2008
Title RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Plus/Minus4-OHT-Sample1-Repl-3
Sample type RNA
 
Channel 1
Source name RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Plus 4-OHT-Sample1-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 RTAdeltaNLS1,2-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-Sample1-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 RTAdeltaNLS1,2-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 RtaΔNLS1,2-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 RtaΔNLS1,2-ER to the dye ratios for ER vector using the software program significance analysis of microarrays (SAM).
 
Submission date May 12, 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) -1.552 0.341036959 0.341036959 10930 35850 110 1718 1699 290 17 1136 1136 1176 504 42 58 5 0 4281 4307 562 13 2574 2574 2758 2048 74 28 0 0 0.341 0.325 0.329 0.319 1.774 0.125 0.225 80 656 100 2289 2296 582 1707 563 1733 135883 344530 1.038 0.756 0 0 0
10_ORF11 -1.321 0.400257405 0.400257405 11920 31340 130 5102 5039 802 15 1350 1350 1485 1637 110 96 77 0 12243 12116 1245 10 2866 2866 3175 2073 65 100 100 0 0.4 0.399 0.41 0.388 1.367 0.388 0.882 120 900 100 13129 12939 3752 9377 3689 9250 604712 1453894 2.171 4.313 0 0 0
100_PAN-AS -0.609 0.655651007 0.655651007 15680 31320 110 2305 2308 308 13 1087 1087 1104 287 25 100 98 0 4400 4365 569 13 2542 2542 2591 528 20 100 91 0 0.656 0.67 0.688 0.682 1.428 0.573 0.698 80 656 100 3076 3044 1218 1858 1221 1823 184602 349218 4.195 3.36 0 0 0
101_UPPAN-A -1.722 0.303128205 0.303128205 15920 35810 120 1548 1546 360 23 995 995 970 271 27 75 50 0 4433 4199 1052 25 2609 2609 2519 629 24 78 70 0 0.303 0.347 0.316 0.315 1.999 0.268 0.507 120 800 100 2377 2141 553 1824 551 1590 185510 503831 2.125 2.671 0 0 0
102_UPPAN-B -1.166 0.445655248 0.445655248 15930 31320 110 5017 5085 597 11 1073 1073 1092 283 25 100 100 0 11190 11249 989 8 2341 2341 2457 603 24 100 100 0 0.446 0.45 0.449 0.448 1.147 0.432 0.895 80 656 100 12793 12920 3944 8849 4012 8908 406764 899946 14.11 14.58 0 0 0
103_UPPAN-C -1.212 0.431669779 0.431669779 16180 35830 100 2162 2143 270 12 979 979 976 228 23 100 100 0 5504 5576 636 11 2763 2763 2705 503 18 100 100 0 0.432 0.414 0.426 0.411 1.324 0.402 0.792 80 560 100 3924 3977 1183 2741 1164 2813 171401 446107 5.118 5.708 0 0 0
104_UPPAN-D -1.279 0.412081042 0.412081042 16180 31320 120 6066 5528 1815 32 1005 1005 1018 198 19 96 93 0 14551 13054 3970 30 2268 2268 2289 337 14 99 97 0 0.412 0.419 0.41 0.386 1.923 0.414 0.926 120 814 100 17344 15309 5061 12283 4523 10786 663310 1566471 22.778 31.944 0 0 0
105_UPPAN-E -1.536 0.34484023 0.34484023 16420 35820 120 7403 6762 2211 32 1017 1017 1014 214 21 93 91 0 21328 19272 6194 32 2812 2812 2797 482 17 95 95 0 0.345 0.349 0.345 0.344 1.686 0.343 0.934 120 808 100 24902 22205 6386 18516 5745 16460 811463 2312586 26.86 34.18 0 0 0
106_UPPAN-F -1.577 0.335178148 0.335178148 16440 31320 110 1437 1486 271 18 1029 1029 1041 200 19 83 50 0 3534 3508 528 15 2317 2317 2317 355 15 91 83 0 0.335 0.384 0.372 0.364 2.499 0.328 0.445 80 656 100 1625 1648 408 1217 457 1191 118914 280649 2.225 3.355 0 0 0
11_K2 -1.152 0.450000965 0.450000965 12180 35850 120 2410 2347 567 24 1097 1097 1146 370 32 94 81 0 5444 5400 921 17 2527 2527 2618 778 29 97 92 0 0.45 0.435 0.415 0.413 1.643 0.42 0.763 120 802 100 4230 4123 1313 2917 1250 2873 281667 647978 3.246 3.576 0 0 0
12_ORF2 -1.384 0.38315499 0.38315499 12160 31330 110 1678 1689 330 19 1299 1299 1340 338 25 61 18 0 3732 3706 556 15 2743 2743 2851 804 28 66 13 0 0.383 0.405 0.413 0.431 2.711 0.297 0.295 80 656 100 1368 1353 379 989 390 963 135105 296477 1.033 1.063 0 0 0
13_K3 -1.472 0.360482218 0.360482218 12430 35850 120 2520 2440 534 21 1190 1190 1364 796 58 83 25 0 6348 6105 1201 19 2658 2658 3249 2509 77 80 4 0 0.36 0.363 0.357 0.357 1.826 0.348 0.756 120 802 100 5020 4697 1330 3690 1250 3447 292766 732606 1.352 1.138 0 0 0
14_ORF70 -1.425 0.372419366 0.372419366 12400 31330 130 3304 3341 773 23 1321 1321 1352 253 18 97 95 0 8205 8218 1798 21 2881 2881 2948 658 22 99 97 0 0.372 0.378 0.372 0.372 1.851 0.356 0.807 120 914 100 7307 7357 1983 5324 2020 5337 400978 986213 7.862 8.009 0 0 0
15_K4 -1.599 0.33010571 0.33010571 12690 35850 110 1961 2288 1653 72 1157 1157 1296 880 67 42 10 0 5106 6194 5737 92 2670 2670 3039 2054 67 67 8 0 0.33 0.321 0.333 0.33 1.845 0.297 0.766 80 656 100 3240 4655 804 2436 1131 3524 183039 495483 1.127 1.536 0 0 0
16_K4_1 -1.079 0.473356816 0.473356816 12670 31330 120 2228 2255 484 21 1406 1406 1458 323 22 89 70 0 4756 4885 1014 20 3020 3020 3102 561 18 94 80 0 0.474 0.455 0.462 0.469 1.931 0.34 0.371 120 814 100 2558 2714 822 1736 849 1865 270618 586226 2.467 3.178 0 0 0
17_K4_2 -1.474 0.35998283 0.35998283 12930 35850 120 4349 4552 2182 47 1147 1147 1357 1412 104 93 66 0 11542 12106 6302 52 2647 2647 3311 4071 122 93 60 0 0.36 0.36 0.359 0.348 1.88 0.31 0.779 120 802 100 12097 12864 3202 8895 3405 9459 546258 1452747 2.263 2.16 0 0 0
18_K5 -1.438 0.369078601 0.369078601 12920 31340 110 2089 2070 434 20 1347 1347 1395 323 23 85 57 0 4955 5121 771 15 2945 2945 3030 568 18 100 95 0 0.369 0.332 0.321 0.302 1.69 0.302 0.445 80 656 100 2752 2899 742 2010 723 2176 165575 409682 2.09 3.681 0 0 0
19_K6 -1.473 0.360232437 0.360232437 13180 35850 120 2301 2239 504 22 1078 1078 1081 219 20 93 86 0 5896 5756 1151 19 2500 2500 2496 456 18 98 96 0 0.36 0.357 0.364 0.327 2.484 0.339 0.775 120 802 100 4619 4417 1223 3396 1161 3256 268659 690722 5.288 7.149 0 0 0
2_K1(subgroupB) -1.493 0.355273011 0.355273011 10910 31340 130 4549 4524 629 13 1360 1360 1515 1824 120 95 25 0 11934 11502 2070 17 2959 2959 3464 4085 117 95 70 0 0.355 0.37 0.361 0.383 1.422 0.435 0.78 120 913 100 12164 11707 3189 8975 3164 8543 542874 1380216 1.65 1.968 0 0 0
20_K7 -0.356 0.78132788 0.78132788 13170 31330 120 5895 5758 1117 19 1371 1371 1410 290 20 99 99 0 8820 8407 1771 21 3029 3029 3138 621 19 97 93 0 0.781 0.816 0.79 0.865 1.566 0.768 0.872 120 792 100 10315 9765 4524 5791 4387 5378 690911 1008803 14.993 8.485 0 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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