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Sample GSM288390 Query DataSets for GSM288390
Status Public on Aug 31, 2008
Title RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Plus/Minus4-OHT-Sample2B-Repl-3
Sample type RNA
 
Channel 1
Source name RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Plus 4-OHT-Sample2B-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 RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Minus 4-OHT-Sample2B-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_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) -1.113 0.462331639 1.032923679 1.410382442 11960 35290 130 4880 4652 888 19 3994 3994 3752 756 20 58 5 0 5884 5738 1027 17 3967 3967 3822 688 18 87 76 0 0.462 0.372 0.474 0.51 2.295 0.798 0.765 120 902 100 2803 2429 886 1917 658 1771 558199 688502 1.19 2.785 0 0 0
10_ORF11 -2.422 0.186597298 0.416888552 0.569231111 12910 30760 150 4054 4135 669 16 3795 3795 3535 718 20 33 1 0 5245 5342 1055 19 3857 3857 3672 692 18 76 50 0 0.187 0.229 0.324 0.285 2.559 0.686 0.638 156 1094 100 1647 1825 259 1388 340 1485 645004 833351 0.836 2.413 0 0 0
100_PAN-AS -3.744 0.074635199 0.16674711 0.227681096 16690 30770 100 2347 2385 507 21 2317 2317 2343 239 10 38 22 0 3843 3987 784 19 3441 3441 3476 387 11 51 36 0 0.075 0.125 0.544 0.613 3.359 0.505 0.616 80 560 100 432 614 30 402 68 546 190787 318990 0.176 1.32 -50 0 0
101_UPPAN-A -2.141 0.226722582 0.506534928 0.691636743 16900 35250 100 2045 2076 289 13 1967 1967 1969 194 9 31 16 0 4063 4177 567 13 3719 3719 3738 375 10 43 22 0 0.227 0.238 0.417 0.445 3.312 0.371 0.402 80 546 100 422 567 78 344 109 458 166116 334177 0.552 1.171 -50 0 0
102_UPPAN-B -1.525 0.347479555 0.776325542 1.060016278 16940 30740 120 4322 4128 896 21 2057 2057 2065 238 11 94 92 0 9780 9382 2335 24 3260 3260 3326 476 14 97 94 0 0.347 0.338 0.345 0.331 1.574 0.343 0.929 120 796 100 8785 8193 2265 6520 2071 6122 495402 1125880 8.668 12.723 0 0 0
103_UPPAN-C 17150 35260 100 1871 1925 384 19 1921 1921 1909 183 9 33 21 0 4417 4551 842 18 4010 4010 4023 391 9 50 42 0 -0.123 0.007 0.406 0.489 2.554 0.359 0.545 80 547 100 357 545 -50 407 4 541 153976 364051 0.087 1.35 -50 0 0
104_UPPAN-D -2.024 0.245875519 0.549325687 0.750064423 17190 30740 100 2542 2571 375 14 1998 1998 2041 351 17 65 42 0 5548 5405 898 16 3336 3336 3466 709 20 88 76 0 0.246 0.277 0.274 0.26 1.604 0.297 0.739 80 560 100 2756 2642 544 2212 573 2069 205713 432401 1.51 2.735 0 0 0
105_UPPAN-E -2.463 0.181369026 0.405207746 0.553281818 17400 35260 100 1983 1972 315 15 1700 1700 1710 185 10 56 42 0 5627 5441 936 17 4067 4067 4097 404 9 78 68 0 0.181 0.198 0.24 0.224 2.378 0.24 0.591 80 560 100 1843 1646 283 1560 272 1374 157742 435312 1.416 3.327 -50 0 0
106_UPPAN-F -2.418 0.187115373 0.418046016 0.570811545 17440 30770 100 2052 2089 271 12 1930 1930 1916 252 13 35 12 0 4025 4024 521 12 3373 3373 3352 345 10 70 48 0 0.187 0.244 0.376 0.366 3.846 0.355 0.409 80 560 100 774 810 122 652 159 651 167144 321919 0.687 1.948 -50 0 0
11_K2 -0.906 0.533662669 1.19228874 1.627983885 13220 35280 140 5516 5271 903 17 3940 3940 3918 328 8 90 85 0 7135 6883 1202 17 4181 4181 4185 423 10 96 93 0 0.534 0.493 0.53 0.502 1.594 0.55 0.838 156 953 100 4530 4033 1576 2954 1331 2702 822273 1073706 4.125 6.378 0 0 0
12_ORF2 -0.981 0.50662845 1.131889921 1.545513675 13150 30770 150 5086 5040 473 9 3873 3873 3796 467 12 89 70 0 6353 6314 637 10 3958 3958 3924 494 12 98 97 0 0.506 0.495 0.495 0.452 1.738 0.545 0.77 156 1021 100 3608 3523 1213 2395 1167 2356 786226 985001 2.664 4.838 0 0 0
13_K3 -1.271 0.414372452 0.925775096 1.264078816 13460 35280 140 4825 4576 795 17 3950 3950 3835 557 14 65 30 0 6387 6043 1135 18 4275 4275 4228 650 15 82 74 0 0.414 0.354 0.43 0.445 2.734 0.489 0.673 156 916 100 2987 2394 875 2112 626 1768 713868 942669 1.33 2.792 0 0 0
14_ORF70 -1.673 0.313600551 0.700634366 0.956665462 13400 30730 120 4802 4619 575 12 3803 3803 3715 487 13 68 50 0 7135 6569 1466 22 3950 3950 3897 512 13 85 78 0 0.314 0.312 0.338 0.324 1.919 0.335 0.736 120 762 100 4184 3435 999 3185 816 2619 554269 788313 1.856 5.219 0 0 0
15_K4 -2.245 0.210953949 0.471305251 0.643533171 13710 35280 170 4303 4421 673 15 3939 3939 3833 507 13 42 22 0 6102 6185 1213 19 4376 4376 4313 524 12 84 66 0 0.211 0.266 0.32 0.273 2.79 0.477 0.606 208 1386 100 2090 2291 364 1726 482 1809 919492 1286564 1.16 3.573 0 0 0
16_K4_1 -0.986 0.504875649 1.127973879 1.540166604 13650 30770 140 5341 5185 750 14 3822 3822 3724 493 13 93 85 0 6974 6873 1029 14 3965 3965 3907 502 12 95 94 0 0.505 0.469 0.493 0.494 1.636 0.609 0.81 156 1002 100 4528 4271 1519 3009 1363 2908 808842 1072211 2.963 5.908 0 0 0
17_K4_2 -1.681 0.311866393 0.696759976 0.951375263 10950 35530 150 4577 4386 911 20 3488 3488 3209 737 22 56 34 0 7063 6586 1636 24 3571 3571 3409 707 20 89 80 0 0.312 0.298 0.353 0.321 2.283 0.48 0.713 156 1081 100 4581 3913 1089 3492 898 3015 684248 1027391 1.597 4.494 0 0 0
18_K5 -1.663 0.315781815 0.705507664 0.963319596 10890 31020 140 5452 5283 792 14 3679 3679 3645 325 8 94 83 0 9430 8962 1827 20 3815 3815 3797 365 9 100 99 0 0.316 0.312 0.317 0.281 1.63 0.314 0.862 156 998 100 7388 6751 1773 5615 1604 5147 824122 1398032 5.04 14.151 0 0 0
19_K6 11200 35510 100 3530 3611 482 13 3553 3553 3431 610 17 15 2 0 4012 4234 749 17 3692 3692 3625 635 17 33 18 0 -0.072 0.107 0.489 0.469 4.274 0.521 0.483 80 546 100 297 600 -23 320 58 542 288850 338737 0.295 0.959 -50 0 0
2_K1(subgroupB) -1.475 0.359733395 0.803702603 1.097397671 11900 30770 140 4911 4731 656 13 3855 3855 3815 566 14 81 39 0 6880 6666 858 12 3944 3944 3889 519 13 96 93 0 0.36 0.322 0.356 0.366 2.122 0.383 0.595 156 968 100 3992 3598 1056 2936 876 2722 738073 1039888 1.618 5.351 0 0 0
20_K7 0.515 1.42899414 3.192604094 4.359269567 11150 31020 150 8758 8893 1096 12 3713 3713 3690 358 9 100 100 0 7386 7233 862 11 3855 3855 3847 510 13 97 97 0 1.429 1.533 1.493 1.585 1.656 1.517 0.815 156 1035 100 8576 8558 5045 3531 5180 3378 1387257 1128345 14.534 6.639 0 0 0

Total number of rows: 107

Table truncated, full table size 28 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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