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Sample GSM288329 Query DataSets for GSM288329
Status Public on Aug 31, 2008
Title RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Plus/Minus4-OHT-Sample2A-Repl-1
Sample type RNA
 
Channel 1
Source name RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Plus 4-OHT-Sample2A-Repl-1
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 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-Sample2A-Repl-1
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 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_GAPDHNorm VALUE_Linear normalized to average of 3 replicates of VALUE_Linear of GAPDH
VALUE VALUE_GAPDHNorm 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_GAPDHNorm 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) -0.692 0.618995145 0.618995145 0.845193021 11000 33840 190 16876 16942 1542 9 12912 12912 12903 1394 10 96 79 0 12834 12926 1341 10 6430 6430 6412 726 11 100 99 0 0.619 0.62 0.619 0.578 1.594 0.727 0.798 256 1509 100 10368 10526 3964 6404 4030 6496 4337239 3309169 2.897 8.972 0 0 0
10_ORF11 -1.708 0.306084098 0.306084098 0.417935658 11980 29310 170 15413 15487 1569 10 10639 10639 10721 862 8 98 97 0 22258 22105 3094 13 6666 6666 6749 927 13 99 99 0 0.306 0.314 0.315 0.3 1.509 0.321 0.886 208 1184 100 20366 20287 4774 15592 4848 15439 3221205 4597751 5.529 16.565 0 0 0
100_PAN-AS -1.363 0.388773018 0.388773018 0.530841387 15750 29290 120 14344 14169 1374 9 12223 12223 12346 935 7 88 60 0 14347 14367 1774 12 8893 8893 8920 756 8 97 96 0 0.389 0.355 0.386 0.361 1.855 0.412 0.555 120 754 100 7575 7420 2121 5454 1946 5474 1700339 1724034 1.95 7.205 0 0 0
101_UPPAN-A -1.058 0.480297432 0.480297432 0.655811343 16000 33800 150 14786 14842 931 6 12554 12554 12627 852 6 96 71 0 12233 12232 928 7 7586 7586 7575 646 8 100 100 0 0.48 0.492 0.503 0.472 1.459 0.556 0.688 156 960 100 6879 6934 2232 4647 2288 4646 2315418 1908217 2.6 7.209 0 0 0
102_UPPAN-B -1.378 0.384751805 0.384751805 0.525350712 16010 29270 170 16724 16831 1687 10 12145 12145 12211 931 7 98 94 0 20682 21008 3954 18 8783 8783 8783 809 9 98 98 0 0.385 0.383 0.377 0.388 1.437 0.378 0.844 208 1316 100 16478 16911 4579 11899 4686 12225 3500822 4369734 4.962 15.111 0 0 0
103_UPPAN-C -0.75 0.594603558 0.594603558 0.811888075 16250 33790 170 15821 15896 1069 6 12586 12586 12681 882 6 100 95 0 13022 13030 1055 8 7581 7581 7569 662 8 99 99 0 0.595 0.607 0.598 0.592 1.366 0.639 0.77 208 1283 100 8676 8759 3235 5441 3310 5449 3306304 2710216 3.645 8.249 0 0 0
104_UPPAN-D -1.774 0.292396916 0.292396916 0.399246803 16240 29300 160 17347 17229 1777 10 12229 12229 12420 1119 9 93 90 0 26222 24657 4825 19 8723 8723 8842 1133 12 96 96 0 0.292 0.314 0.309 0.321 1.595 0.296 0.808 208 1056 100 22617 20934 5118 17499 5000 15934 3583718 5128599 4.298 13.959 0 0 0
105_UPPAN-E -1.667 0.314907495 0.314907495 0.429983367 16490 33790 150 17624 17607 1003 5 12371 12371 12286 1379 11 99 98 0 24057 23948 1828 7 7376 7376 7316 976 13 100 100 0 0.315 0.316 0.319 0.311 1.246 0.345 0.772 156 1010 100 21934 21808 5253 16681 5236 16572 2746658 3735928 3.859 17.041 0 0 0
106_UPPAN-F -1.692 0.309497573 0.309497573 0.422596511 16480 29260 160 13235 13377 1166 8 11879 11879 12017 1046 8 64 29 0 12698 12534 1706 13 8316 8316 8354 871 10 93 90 0 0.309 0.355 0.352 0.32 2.289 0.439 0.346 208 1255 100 5738 5716 1356 4382 1498 4218 2782435 2606974 1.3 4.799 0 0 0
11_K2 -0.687 0.621144141 0.621144141 0.848127319 12260 33830 190 16480 16309 2268 13 11549 11549 11811 1117 9 94 88 0 13945 13779 1938 14 6008 6008 6067 623 10 98 98 0 0.621 0.613 0.624 0.596 1.586 0.643 0.778 256 1260 100 12868 12531 4931 7937 4760 7771 4175115 3527449 4.027 12.379 0 0 0
12_ORF2 -2.581 0.167125062 0.167125062 0.228197163 12240 29290 160 11674 11530 1113 9 11122 11122 11102 1130 10 27 2 0 10228 10067 1513 15 6924 6924 6951 950 13 88 83 0 0.167 0.13 0.303 0.282 2.389 0.461 0.21 208 1210 100 3856 3551 552 3304 408 3143 2398216 2093900 0.379 3.28 0 0 0
13_K3 -1.291 0.408667664 0.408667664 0.558006085 12510 33820 240 16017 16003 1746 10 12087 12087 12091 1305 10 94 82 0 15862 15760 2117 13 6245 6245 6261 688 10 99 99 0 0.409 0.412 0.41 0.39 1.561 0.438 0.747 460 1870 100 13547 13431 3930 9617 3916 9515 7361295 7249554 2.998 13.807 0 0 0
14_ORF70 -2.331 0.198746313 0.198746313 0.27137369 12490 29300 160 13416 13009 1832 14 11410 11410 11405 1187 10 71 37 0 16978 16398 2677 16 6882 6882 6890 771 11 98 98 0 0.199 0.168 0.214 0.188 2.13 0.257 0.445 208 1036 100 12102 11115 2006 10096 1599 9516 2705868 3410760 1.351 12.332 0 0 0
15_K4 -0.843 0.557483109 0.557483109 0.761202793 12760 33820 180 16309 16299 1154 7 12973 12973 12946 1321 10 96 77 0 12641 12594 887 7 6659 6659 6657 750 11 100 100 0 0.558 0.56 0.571 0.547 1.353 0.689 0.686 256 1113 100 9318 9261 3336 5982 3326 5935 4172656 3224046 2.538 7.916 0 0 0
16_K4_1 -1.072 0.475659138 0.475659138 0.649478089 12740 29300 180 13960 13607 1622 11 11460 11460 11634 1086 9 79 58 0 12082 11998 2035 16 6827 6827 6928 814 11 96 93 0 0.476 0.415 0.466 0.425 1.876 0.484 0.562 256 1358 100 7755 7318 2500 5255 2147 5171 3483334 3071482 1.817 6.229 0 0 0
17_K4_2 -1.288 0.409518349 0.409518349 0.559167633 13010 33820 150 18641 18496 2121 11 13563 13563 13604 1304 9 96 80 0 19378 19784 2667 13 6982 6982 6943 757 10 100 100 0 0.41 0.385 0.397 0.347 1.565 0.433 0.748 156 910 100 17474 17735 5078 12396 4933 12802 2885298 3086369 3.752 16.963 0 0 0
18_K5 -1.774 0.292396916 0.292396916 0.399246803 12990 29310 110 14057 13680 1640 11 11106 11106 11297 992 8 83 70 0 16686 16478 2563 15 6594 6594 6746 889 13 100 100 0 0.292 0.26 0.302 0.232 2.113 0.241 0.407 80 589 100 13043 12458 2951 10092 2574 9884 1094362 1318251 2.402 10.947 0 0 0
19_K6 -1.291 0.408667664 0.408667664 0.558006085 13260 33820 180 16647 16496 1555 9 13875 13875 13939 1337 9 82 54 0 13944 13757 1416 10 7160 7160 7156 815 11 100 99 0 0.409 0.397 0.421 0.369 1.938 0.504 0.614 256 1217 100 9556 9218 2772 6784 2621 6597 4222946 3521776 1.912 8.099 0 0 0
2_K1(subgroupB) -1.886 0.270556161 0.270556161 0.369424835 10990 29320 180 16064 15573 2118 13 11416 11416 11426 1219 10 88 83 0 24060 22792 4726 20 6878 6878 6904 781 11 96 95 0 0.271 0.261 0.276 0.257 1.818 0.279 0.79 256 1443 100 21830 20071 4648 17182 4157 15914 3986581 5834693 3.402 20.343 0 0 0
20_K7 -0.967 0.511568735 0.511568735 0.698510041 13230 29290 180 18249 18569 3358 18 10723 10723 10738 1661 15 94 91 0 20907 19972 4481 22 6194 6194 6283 1128 17 96 92 0 0.512 0.569 0.531 0.582 1.724 0.571 0.835 256 1396 100 22239 21624 7526 14713 7846 13778 4753573 5112844 4.715 12.136 0 0 0

Total number of rows: 107

Table truncated, full table size 30 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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