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Sample GSM288336 Query DataSets for GSM288336
Status Public on Aug 31, 2008
Title VectorTransfected-Vero-rKSHV.219cells-Hygro-Plus/Minus4-OHT-Sample2A-Repl-1
Sample type RNA
 
Channel 1
Source name Vector-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 Vector-Transfected-Vero-rKSHV.219cells-Hygro-Minus 4-OHT-Sample2A-Repl-1
Organism 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) -1.786 0.289974914 0.720254815 0.720254815 10990 33950 190 14678 14557 1207 8 13293 13293 13218 1078 8 60 23 0 15336 15209 1314 8 10561 10561 10530 982 9 98 97 0 0.29 0.272 0.333 0.262 2.334 0.377 0.395 256 1596 100 6160 5912 1385 4775 1264 4648 3726591 3893617 1.242 4.765 0 0 0
10_ORF11 -2.317 0.200684347 0.498470249 0.498470249 11980 29450 170 18256 18175 1249 6 15185 15185 15125 1040 6 94 85 0 27407 27293 1972 7 12109 12109 12113 1010 8 100 100 0 0.201 0.197 0.201 0.184 1.688 0.216 0.677 208 1232 100 18369 18174 3071 15298 2990 15184 3780458 5676958 2.933 15.03 0 0 0
100_PAN-AS 15740 29430 140 13935 13745 1263 9 14611 14611 14403 1450 10 0 0 0 15837 15615 1833 11 12735 12735 12631 1243 9 80 63 0 -0.218 -0.301 0.127 0.191 7.915 0.549 0.213 156 982 100 2426 2014 -676 3102 -866 2880 2144280 2435915 -0.454 2.401 0 0 0
101_UPPAN-A 15990 33930 130 13276 13212 1085 8 13429 13429 13169 1303 9 8 0 0 14703 14608 1305 8 11709 11709 11549 1240 10 93 67 0 -0.051 -0.075 0.158 0.153 5.303 0.37 0.15 120 810 100 2841 2682 -153 2994 -217 2899 1585486 1752916 0.033 2.467 0 0 0
102_UPPAN-B -3.059 0.119991158 0.298040296 0.298040296 15990 29430 140 14789 14658 1113 7 14073 14073 13972 1331 9 28 0 0 18378 18289 1390 7 12411 12411 12358 1155 9 100 99 0 0.12 0.1 0.168 0.13 2.927 0.224 0.211 156 998 100 6683 6463 716 5967 585 5878 2286611 2853071 0.515 5.135 0 0 0
103_UPPAN-C 16240 33930 160 12938 12615 1428 11 13264 13264 12972 1528 11 1 0 0 14501 14176 1729 12 11710 11710 11525 1392 12 75 50 0 -0.117 -0.263 0.215 0.255 6.725 0.664 0.223 208 1183 100 2465 1817 -326 2791 -649 2466 2624019 2948647 -0.234 1.904 0 0 0
104_UPPAN-D -4.187 0.054901904 0.136368213 0.136368213 16240 29430 130 14906 14540 1395 9 14169 14169 14148 1366 9 24 0 0 26052 25905 1946 7 12626 12626 12630 1202 9 100 100 0 0.055 0.028 0.073 0.059 2.794 0.082 0.121 120 882 100 14163 13650 737 13426 371 13279 1744788 3108635 0.287 11.044 0 0 0
105_UPPAN-E -3.825 0.070560275 0.175261292 0.175261292 16490 33930 160 14207 14102 898 6 13266 13266 13084 1380 10 27 0 0 25174 23962 4460 18 11839 11839 11762 1347 11 94 92 0 0.071 0.069 0.085 0.08 3.147 0.076 0.145 208 1066 100 14276 12959 941 13335 836 12123 2933289 4984140 0.738 9.057 0 0 0
106_UPPAN-F 16490 29420 110 14054 13694 1564 11 14813 14813 14619 1499 10 0 0 0 16053 15700 1737 11 13341 13341 13144 1342 10 80 51 0 -0.28 -0.474 0.104 0.235 9.633 0.564 0.222 80 656 100 1953 1240 -759 2712 -1119 2359 1095495 1256036 -0.617 1.905 0 0 0
11_K2 -1.826 0.282045534 0.700559409 0.700559409 12240 33960 200 13925 13729 1526 11 12062 12062 12082 1108 9 66 42 0 16195 16097 1799 11 9591 9591 9607 966 10 99 97 0 0.282 0.256 0.305 0.27 1.829 0.353 0.473 316 1375 100 8467 8173 1863 6604 1667 6506 4338395 5086656 1.486 6.718 0 0 0
12_ORF2 12240 29450 150 13465 13009 2180 16 14383 14383 14190 1496 10 1 0 0 14190 13804 2106 15 11488 11488 11391 1270 11 76 56 0 -0.34 -0.593 0.186 0.322 8.614 0.905 0.354 156 984 100 1784 942 -918 2702 -1374 2316 2029368 2153497 -0.789 1.9 0 0 0
13_K3 -3.108 0.115984185 0.288087567 0.288087567 12470 33960 220 13312 13120 1365 10 12297 12297 12252 988 8 52 17 0 18631 18014 2274 12 9878 9878 9952 1429 14 97 95 0 0.116 0.101 0.148 0.131 2.233 0.17 0.371 392 1596 100 9768 8959 1015 8753 823 8136 5142932 7061331 0.879 5.642 0 0 0
14_ORF70 -3.211 0.107992273 0.268236842 0.268236842 12480 29450 170 15496 14764 2287 15 14434 14434 14277 1450 10 34 0 0 21512 20879 3032 14 11677 11677 11539 1218 10 97 96 0 0.108 0.036 0.128 0.114 2.323 0.211 0.232 208 1142 100 10897 9532 1062 9835 330 9202 3070812 4342759 0.336 7.668 0 0 0
15_K4 -2.126 0.229092161 0.569031059 0.569031059 12740 33950 150 13852 13627 1288 9 12940 12940 12991 898 6 50 15 0 14661 14528 1304 8 10680 10680 10765 1563 14 96 79 0 0.229 0.179 0.281 0.264 2.662 0.26 0.285 156 996 100 4893 4535 912 3981 687 3848 2125834 2266360 0.708 2.408 0 0 0
16_K4_1 -2.594 0.165625877 0.411390192 0.411390192 12730 29450 170 15554 15343 1538 10 14995 14995 14983 1199 8 29 2 0 15636 15518 1359 8 12260 12260 12213 1008 8 93 86 0 0.166 0.107 0.277 0.245 2.34 0.489 0.268 208 1296 100 3935 3606 559 3376 348 3258 3191252 3227730 0.3 3.279 0 0 0
17_K4_2 -2.774 0.146198458 0.363135356 0.363135356 12990 33950 150 14392 14163 1279 9 12895 12895 12849 1128 8 62 17 0 20823 20501 2756 13 10586 10586 10532 1131 10 97 96 0 0.146 0.128 0.158 0.141 1.881 0.187 0.354 156 1002 100 11734 11183 1497 10237 1268 9915 2209437 3198097 1.165 8.814 0 0 0
18_K5 -3.624 0.081108673 0.201461952 0.201461952 12980 29440 110 15492 15197 1364 8 15189 15189 15135 1354 8 12 0 0 16195 15979 1498 9 12458 12458 12388 1205 9 90 77 0 0.081 0.002 0.214 0.159 2.752 0.33 0.135 80 618 100 4040 3529 303 3737 8 3521 1215739 1278293 0.046 2.98 0 0 0
19_K6 -2.927 0.131487722 0.326596063 0.326596063 13240 33960 170 13148 13065 966 7 12429 12429 12389 980 7 40 6 0 15789 15637 1309 8 10322 10322 10279 1052 10 100 99 0 0.132 0.12 0.168 0.139 2.592 0.244 0.281 208 1335 100 6186 5951 719 5467 636 5315 2717507 3252511 0.69 5.093 0 0 0
2_K1(subgroupB) -3.214 0.107767943 0.267679638 0.267679638 10990 29460 180 16451 16119 1565 9 14656 14656 14647 1042 7 67 42 0 28188 26839 3985 14 11536 11536 11565 993 8 98 98 0 0.108 0.096 0.122 0.103 1.984 0.136 0.47 256 1464 100 18447 16766 1795 16652 1463 15303 4126347 6870764 1.413 15.382 0 0 0
20_K7 -2.933 0.130942016 0.325240609 0.325240609 13230 29450 180 16054 16165 1188 7 14835 14835 14826 1098 7 55 24 0 21400 21293 2010 9 12091 12091 12061 1061 8 99 99 0 0.131 0.145 0.156 0.138 2.149 0.176 0.373 256 1334 100 10528 10532 1219 9309 1330 9202 4138242 5451059 1.219 8.701 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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