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Sample GSM288331 Query DataSets for GSM288331
Status Public on Aug 31, 2008
Title RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Plus/Minus4-OHT-Sample2A-Repl-2
Sample type RNA
 
Channel 1
Source name RTAdeltaNLS1,2-ER-Transfected-Vero-rKSHV.219cells-Hygro-Plus 4-OHT-Sample1-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-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 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) -0.449 0.732550437 0.732550437 1.000244544 11030 34840 190 17316 16959 2694 15 13363 13363 13557 1494 11 79 62 0 11644 11100 2348 21 6246 6246 6458 1242 19 88 82 0 0.732 0.741 0.772 0.727 2.09 0.93 0.77 256 1599 100 9351 8450 3953 5398 3596 4854 4341408 2841697 2.277 3.738 0 0 0
10_ORF11 -1.483 0.35774413 0.35774413 0.488473689 12000 30290 140 14524 14229 2572 18 9232 9232 9154 1514 16 89 83 0 20317 19098 5282 27 5524 5524 5558 930 16 93 93 0 0.358 0.368 0.377 0.39 1.795 0.406 0.859 156 1002 100 20085 18571 5292 14793 4997 13574 2219667 2979292 3.352 14.559 0 0 0
100_PAN-AS -0.678 0.625031151 0.625031151 0.85343475 15760 30310 130 15463 15417 1366 8 12323 12323 12437 956 7 96 80 0 13643 13743 1739 12 8619 8619 8648 870 10 100 100 0 0.625 0.604 0.58 0.568 1.616 0.659 0.748 120 877 100 8164 8218 3140 5024 3094 5124 1850005 1649200 3.117 5.856 0 0 0
101_UPPAN-A -0.433 0.740719899 0.740719899 1.011399352 16020 34790 110 16351 16201 1571 9 12943 12943 13055 978 7 97 88 0 12122 12111 1077 8 7521 7521 7533 673 8 98 97 0 0.741 0.71 0.731 0.715 1.437 0.863 0.743 80 656 100 8009 7848 3408 4601 3258 4590 1296057 968857 3.217 6.802 0 0 0
102_UPPAN-B -1.097 0.467487599 0.467487599 0.638320444 16030 30280 140 17875 17469 2291 13 12374 12374 12455 848 6 91 89 0 20364 20757 4233 20 8595 8595 8691 1053 12 95 94 0 0.467 0.419 0.439 0.429 1.719 0.442 0.878 156 1022 100 17270 17257 5501 11769 5095 12162 2725148 3238162 5.913 11.459 0 0 0
103_UPPAN-C -0.199 0.871154192 0.871154192 1.189497929 16280 34760 160 17133 16857 1857 11 12583 12583 12673 850 6 91 89 0 12570 12439 1903 15 7347 7347 7365 657 8 95 92 0 0.871 0.839 0.835 0.809 1.477 0.893 0.852 208 1183 100 9773 9366 4550 5223 4274 5092 3506212 2587319 4.922 7.723 0 0 0
104_UPPAN-D -1.609 0.327825504 0.327825504 0.447621972 16270 30280 140 17416 17145 2018 11 12281 12281 12511 1308 10 91 86 0 24265 23270 4264 18 8601 8601 9285 3141 33 94 92 0 0.328 0.332 0.335 0.305 1.954 0.348 0.866 156 906 100 20799 19533 5135 15664 4864 14669 2674577 3630068 3.543 4.452 0 0 0
105_UPPAN-E -1.334 0.396666922 0.396666922 0.541619941 16530 34790 150 19049 18886 1384 7 12393 12393 12564 1512 12 98 97 0 24077 23918 2393 10 7296 7296 7958 3106 39 99 99 0 0.397 0.391 0.394 0.389 1.255 0.411 0.879 156 967 100 23437 23115 6656 16781 6493 16622 2946224 3731247 4.181 5.138 0 0 0
106_UPPAN-F -1.062 0.478967609 0.478967609 0.653995566 16500 30270 110 13793 13920 1173 8 11996 11996 12112 1019 8 82 37 0 11927 12150 1298 10 8176 8176 8345 1588 19 98 72 0 0.479 0.484 0.457 0.424 2.072 0.595 0.58 80 642 100 5548 5898 1797 3751 1924 3974 1113600 971978 1.774 2.396 0 0 0
11_K2 -1.297 0.406971593 0.406971593 0.555690222 12240 34820 130 13411 12561 3361 26 11078 11078 11081 1367 12 60 43 0 11342 10551 2405 22 5608 5608 5751 1102 19 91 87 0 0.407 0.3 0.531 0.513 2.618 0.779 0.554 120 895 100 8067 6426 2333 5734 1483 4943 1507330 1266102 1.083 4.356 0 0 0
12_ORF2 -0.928 0.525586455 0.525586455 0.717650222 12250 30300 120 12512 12100 1790 14 10279 10279 9744 1742 17 55 13 0 10518 10223 1668 16 6269 6269 6028 1005 16 92 88 0 0.526 0.461 0.55 0.472 1.729 0.81 0.668 120 749 100 6482 5775 2233 4249 1821 3954 1452040 1226760 1.352 4.174 0 0 0
13_K3 -2.549 0.170873431 0.170873431 0.23331529 12490 34820 90 13616 13200 3894 29 12690 12690 12980 1716 13 46 25 0 11789 11451 2952 25 6371 6371 6861 1849 26 90 76 0 0.171 0.1 0.537 0.54 2.78 0.795 0.359 52 420 100 6344 5590 926 5418 510 5080 686382 595434 0.128 2.482 0 0 0
14_ORF70 -1.494 0.35502684 0.35502684 0.484763426 12490 30320 140 15944 16220 2055 12 11505 11505 11447 1074 9 94 92 0 19306 19213 2351 12 6801 6801 6774 731 10 100 100 0 0.355 0.38 0.375 0.351 1.639 0.409 0.812 156 974 100 16944 17127 4439 12505 4715 12412 2530251 2997185 4.444 17.016 0 0 0
15_K4 -0.379 0.768970416 0.768970416 1.049973386 12750 34790 140 18264 17984 1951 10 13571 13571 13761 1347 9 91 81 0 13003 12740 1861 14 6899 6899 7002 1038 14 95 94 0 0.769 0.756 0.757 0.726 1.531 0.814 0.783 156 1018 100 10797 10254 4693 6104 4413 5841 2805487 1987390 3.135 5.528 0 0 0
16_K4_1 -0.647 0.63860688 0.63860688 0.871971423 12750 30300 170 14873 14694 1561 10 11419 11419 11505 1053 9 90 80 0 12174 11872 1910 16 6766 6766 6786 724 10 94 94 0 0.639 0.641 0.628 0.628 1.406 0.724 0.735 208 1244 100 8862 8381 3454 5408 3275 5106 3056446 2469452 3.028 7.025 0 0 0
17_K4_2 -1.084 0.471719125 0.471719125 0.644098287 13020 34810 140 19508 19121 2681 14 13671 13671 13945 1623 11 89 85 0 19414 19549 2857 14 7039 7039 7781 3865 49 99 94 0 0.472 0.436 0.464 0.414 1.801 0.471 0.785 156 961 100 18212 17960 5837 12375 5450 12510 2982923 3049681 3.189 3.045 0 0 0
18_K5 -1.367 0.387696603 0.387696603 0.529371621 12990 30300 120 13827 13440 2012 14 10736 10736 10753 1066 9 80 63 0 14273 13870 3403 24 6301 6301 6353 825 12 91 90 0 0.388 0.357 0.418 0.37 1.895 0.434 0.766 120 743 100 11063 10273 3091 7972 2704 7569 1612787 1664446 2.521 9.112 0 0 0
19_K6 -0.602 0.658839976 0.658839976 0.89959825 13240 34800 140 18176 18079 2310 12 13156 13156 13361 1585 11 91 76 0 14399 14356 2103 14 6778 6778 7172 2510 34 98 89 0 0.659 0.65 0.65 0.594 1.568 0.73 0.764 156 920 100 12641 12501 5020 7621 4923 7578 2820316 2239472 2.977 2.862 0 0 0
2_K1(subgroupB) -1.448 0.366529189 0.366529189 0.500469051 11010 30300 160 16403 15837 2389 15 10957 10957 10901 919 8 89 85 0 21176 19592 5686 29 6314 6314 6349 782 12 89 88 0 0.366 0.368 0.378 0.409 2.11 0.387 0.874 208 1004 100 20308 18158 5446 14862 4880 13278 3294141 4075152 5.371 16.935 0 0 0
20_K7 -0.933 0.523768064 0.523768064 0.715167341 13250 30300 160 17028 17050 2957 17 10642 10642 10678 870 8 94 92 0 18291 17478 3949 22 6102 6102 6112 678 11 94 94 0 0.524 0.563 0.554 0.563 1.654 0.586 0.883 208 1058 100 18575 17784 6386 12189 6408 11376 3546415 3635485 7.324 16.764 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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