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Sample GSM886339 Query DataSets for GSM886339
Status Public on Mar 07, 2012
Title MTB1254 Day 0 vs low Oxygen Day 4 rep 6
Sample type RNA
 
Channel 1
Source name MTB1254 Day 0
Organism Mycobacterium tuberculosis
Characteristics treatment: control
strain name: H37Rv (wild_type)
Treatment protocol Cell pellets were suspended in 1 ml TRIzol reagent (GIBCO BRL) and transferred to 2-ml screw cap tubes containing 0.5 ml 0.1-mm diameter zirconia/silica beads (BioSpec Products).
Growth protocol Clinical isolate 1254, 7H9 medium (supplemented with BSA, NaCl, glucose, and glycerol), 250-ml vented tissue culture flasks, 90 rpm shaking, and a starting culture density of OD 0.15 were used, unless otherwise indicated. RNA samples isolated from OD 0.15 cultures of the M. tuberculosis strain being assayed on the same day were used for the reference sample in each experiment. Cells were collected with a 4-min centrifugation step and frozen on dry ice.
Extracted molecule total RNA
Extraction protocol Cell debris was separated by a 45-s centrifugation. The supernatant was transferred to 2-ml Heavy Phase Lock Gel I tubes (Eppendorf) containing 300 ul chloroform, inverted rapidly for 15 s, and incubated 2 min. Samples were centrifuged for 5 min, and the aqueous phase was added to 270 ul isopropanol, followed by addition of 270 ul of the following mixture: 0.8 M sodium citrate and 1.2 M NaCl. Samples were incubated for 10 min at 4 C and centrifuged for 15 min at 4 C. The RNA pellets were washed with 1 ml 75% ethanol, centrifuged 5 min, and air-dried. After suspension of the RNA pellets in 90 ul water, 10 ul DNase I 10x buffer, and 6 U DNase I (Ambion) were added, and the samples were incubated for 30 min. Final purification of RNA was by RNeasy column (QIAGEN).
Label Cy3
Label protocol Both a PCR gene product microarray and a 70-mer oligonucleotide-based microarray (tuberculosis oligonucleotide set; QIAGEN) were used. Labeled cDNA was prepared as follows: 2 ug total RNA and 4.4ug of random oligonucleotide hexamers were incubated for 2 min at 98 C, cooled on ice, combined with Stratascript RTase buffer, 0.5 mM dA,G,CTP, 0.02 mM dTTP, 1.5 nmol Cy3 or Cy5-dUTP (Amersham Biosciences), and 1.8 ul Stratascript RTase (Stratagene) in a total volume of 25 ul, and incubated for 10 min at 25 C and 90 min at 42 C. cDNA was purified by microcon-10 (Amicon) filtration.
 
Channel 2
Source name MTB1254 Day 4
Organism Mycobacterium tuberculosis
Characteristics treatment: NRP (Wayne Growth)/4 days
strain name: H37Rv (wild_type)
Treatment protocol Cell pellets were suspended in 1 ml TRIzol reagent (GIBCO BRL) and transferred to 2-ml screw cap tubes containing 0.5 ml 0.1-mm diameter zirconia/silica beads (BioSpec Products).
Growth protocol Clinical isolate 1254, 7H9 medium (supplemented with BSA, NaCl, glucose, and glycerol), 250-ml vented tissue culture flasks, 90 rpm shaking, and a starting culture density of OD 0.15 were used, unless otherwise indicated. RNA samples isolated from OD 0.15 cultures of the M. tuberculosis strain being assayed on the same day were used for the reference sample in each experiment. Cells were collected with a 4-min centrifugation step and frozen on dry ice.
Extracted molecule total RNA
Extraction protocol Cell debris was separated by a 45-s centrifugation. The supernatant was transferred to 2-ml Heavy Phase Lock Gel I tubes (Eppendorf) containing 300 ul chloroform, inverted rapidly for 15 s, and incubated 2 min. Samples were centrifuged for 5 min, and the aqueous phase was added to 270 ul isopropanol, followed by addition of 270 ul of the following mixture: 0.8 M sodium citrate and 1.2 M NaCl. Samples were incubated for 10 min at 4 C and centrifuged for 15 min at 4 C. The RNA pellets were washed with 1 ml 75% ethanol, centrifuged 5 min, and air-dried. After suspension of the RNA pellets in 90 ul water, 10 ul DNase I 10x buffer, and 6 U DNase I (Ambion) were added, and the samples were incubated for 30 min. Final purification of RNA was by RNeasy column (QIAGEN).
Label Cy5
Label protocol Both a PCR gene product microarray and a 70-mer oligonucleotide-based microarray (tuberculosis oligonucleotide set; QIAGEN) were used. Labeled cDNA was prepared as follows: 2 ug total RNA and 4.4ug of random oligonucleotide hexamers were incubated for 2 min at 98 C, cooled on ice, combined with Stratascript RTase buffer, 0.5 mM dA,G,CTP, 0.02 mM dTTP, 1.5 nmol Cy3 or Cy5-dUTP (Amersham Biosciences), and 1.8 ul Stratascript RTase (Stratagene) in a total volume of 25 ul, and incubated for 10 min at 25 C and 90 min at 42 C. cDNA was purified by microcon-10 (Amicon) filtration.
 
 
Hybridization protocol 10 ul of hybridization solution (labeled cDNA, 5 ug tRNA, 3.8x SSC, 0.27% SDS) was sealed under a coverslip with rubber cement and hybridized overnight at 65 C for the DNA microarray. Oligonucleotide microarrays were first prehybridized for 1 h in 5x SSC, 1% BSA, and 0.1% SDS and washed with H2O and isopropanol. After the prehybridization, 10 ul of hybridization solution (labeled cDNA, 5 ug tRNA, 2x SSC, 25% formamide, and 0.1% SDS) was hybridized overnight at 54 C.
Scan protocol Microarrays were scanned using a GenePix 4000A (Axon Instruments, Inc.). The intensities of the two dyes at each spot were quantified using ScanAlyze (M. Eisen, Lawrence Berkeley National Lab, Berkeley, CA).
Description Simple annotation: Strain Comparison, Stress by growth condition
Image: http://smd.stanford.edu/MicroArray/gifs/2007-06/75059.gif
Data processing VALUE is Log (base 2) of the ratio of the mean of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm)
 
Submission date Mar 05, 2012
Last update date Mar 07, 2012
Contact name SMD Staff
E-mail(s) array@genome.stanford.edu
Phone 650-498-6012
URL http://genome-www5.stanford.edu/
Organization name Stanford Microarray Database (SMD)
Department Stanford University, School of Medicine
Street address 300 Pasteur Drive
City Stanford
State/province CA
ZIP/Postal code 94305
Country USA
 
Platform ID GPL9901
Series (1)
GSE8839 Inhibition of Respiration by Nitric Oxide Induces a Mycobacterium tuberculosis Dormancy Program

Data table header descriptions
ID_REF ID_REF
CH1I_MEAN Uncorrected Cy3 mean pixel intensity.; Type: integer; Scale: linear_scale; Channel: Cy3 Channel
CH2I_MEAN Uncorrected Cy3 mean pixel intensity.; Type: integer; Scale: linear_scale; Channel: Cy5 channel
CH1B_MEDIAN Median intensities of background pixels of Cy3.; Type: integer; Scale: linear_scale; Channel: Cy3 Channel
CH2B_MEDIAN Median intensities of background pixels of Cy3.; Type: integer; Scale: linear_scale; Channel: Cy5 channel
CH1D_MEAN The mean feature pixel intensity with the median background subtracted (channel 1).; Type: integer; Scale: linear_scale; Channel: Cy3 Channel
CH2D_MEAN The mean feature pixel intensity with the median background subtracted (channel 2).; Type: integer; Scale: linear_scale; Channel: Cy5 channel
CH1B_MEAN Mean intensities of background pixels of Cy3.; Type: integer; Scale: linear_scale; Background
CH2B_MEAN Mean intensities of background pixels of Cy5.; Type: integer; Scale: linear_scale; Background
PERGTBCH1I_1SD The percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 532 nm.; Type: integer; Scale: linear_scale
PERGTBCH2I_1SD The percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 635 nm.; Type: integer; Scale: linear_scale
PIX_RAT2_MEDIAN Contains median of Ch2PI-CH2B/Ch1PI-CH1B where Ch1PI & Ch2PI represent single pixel intensities.; Type: float; Scale: linear_scale
TOT_SPIX Count of the number of pixels in the spot.; Type: integer; Scale: linear_scale
TOT_BPIX Number of background pixels.; Type: integer; Scale: linear_scale
REGR The regression ratio of every pixel in a 2-feature-diameter circle around the center of the feature.; Type: float; Scale: linear_scale
CORR The correlation between channel1 (Cy3) & Channel 2 (Cy5) pixels within the spot, and is a useful quality control parameter. Generally, high values imply better fit & good spot quality.; Type: float; Scale: linear_scale
TOP Box top: int(((centerX - radius) - Xoffset) / pixelSize).; Type: integer; Scale: linear_scale
BOT Box bottom: int(((centerX + radius) - Xoffset) / pixelSize).; Type: integer; Scale: linear_scale
LEFT Box left: int(((centerY - radius) - yoffset) / pixelSize).; Type: integer; Scale: linear_scale
RIGHT Box right: int(((centerY + radius) - yoffset) / pixelSize); Type: integer; Scale: linear_scale
FLAG User defined spot flag (default 0).; Type: integer; Scale: linear_scale
CH2IN_MEAN Normalized value of mean Channel 2 (usually 635 nm) intensity (CH2I_MEAN/Normalization factor).; Type: integer; Scale: linear_scale; Channel: Cy5 channel
CH2BN_MEDIAN Normalized value of median Channel 2 (usually 635 nm) background (CH2B_MEDIAN/Normalization factor).; Type: integer; Scale: linear_scale; Channel: Cy5 channel; Background
CH2DN_MEAN Normalized value of mean Channel 2 (usually 635 nm) intensity with normalized background subtracted (CH2IN_MEAN - CH2BN_MEDIAN).; Type: integer; Scale: linear_scale; Channel: Cy5 channel
RAT2N_MEAN Type: float; Scale: linear_scale
RAT1N_MEAN Ratio of the means of Channel 1 (usually 532 nm) intensity to normalized Channel 2 (usually 635 nm) intensity with median background subtracted (CH1D_MEAN/CH2DN_MEAN). Channel 1/Channel 2 ratio normalized or Green/Red ratio normalized.; Type: float; Scale: linear_scale
VALUE Log (base 2) of the ratio of the mean of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm) [log (base 2) (RAT2N_MEAN)].; Type: float; Scale: log_base_2

Data table
ID_REF CH1I_MEAN CH2I_MEAN CH1B_MEDIAN CH2B_MEDIAN CH1D_MEAN CH2D_MEAN CH1B_MEAN CH2B_MEAN PERGTBCH1I_1SD PERGTBCH2I_1SD PIX_RAT2_MEDIAN TOT_SPIX TOT_BPIX REGR CORR TOP BOT LEFT RIGHT FLAG CH2IN_MEAN CH2BN_MEDIAN CH2DN_MEAN RAT2N_MEAN RAT1N_MEAN VALUE
1 1718 1503 400 329 1318 1174 null null 77 79 .887 256 1014 .886 .975 68 86 80 98 0 1744 382 1362 1.034 .968 .048
2 1020 865 400 315 620 550 null null 69 71 .866 256 1000 .848 .944 67 85 104 122 0 1004 366 638 1.029 .972 .042
3 2094 1720 394 312 1700 1408 null null 73 77 .847 256 973 .818 .981 67 85 128 146 0 1996 362 1634 .961 1.041 -.057
4 999 807 393 318 606 489 null null 66 70 .798 256 978 .767 .942 65 83 152 170 0 936 369 567 .936 1.068 -.095
5 2302 2128 395 318 1907 1810 null null 76 78 .961 256 993 .94 .985 64 82 176 194 0 2469 369 2100 1.101 .908 .139
6 1195 930 395 326 800 604 null null 73 73 .766 256 946 .743 .953 66 84 199 217 0 1079 378 701 .876 1.141 -.191
7 731 525 398 322 333 203 null null 64 56 .638 256 990 .564 .798 64 82 223 241 0 609 374 236 .707 1.414 -.5
8 1405 1664 400 320 1005 1344 null null 72 77 1.322 256 962 1.308 .975 64 82 247 265 0 1931 371 1559 1.552 .644 .634
9 753 610 395 304 358 306 null null 69 69 .85 256 987 .805 .861 63 81 271 289 0 708 353 355 .992 1.008 -.012
10 822 808 396 309 426 499 null null 63 70 1.212 256 987 1.124 .93 63 81 295 313 0 938 359 579 1.359 .736 .443
11 834 596 398 323 436 273 null null 66 63 .622 256 991 .587 .859 63 81 319 337 0 692 375 317 .727 1.376 -.461
12 728 556 398 312 330 244 null null 65 61 .763 256 978 .691 .839 63 81 343 361 0 645 362 283 .858 1.166 -.221
13 1337 1219 401 305 936 914 null null 76 81 1.004 256 976 .93 .965 62 80 367 385 0 1414 354 1061 1.133 .883 .18
14 707 553 406 317 301 236 null null 64 64 .808 256 966 .698 .833 62 80 391 409 0 642 368 274 .91 1.099 -.136
15 2030 2130 415 323 1615 1807 null null 75 80 1.122 256 955 1.112 .985 62 80 415 433 0 2472 375 2097 1.298 .77 .377
16 2679 2970 423 328 2256 2642 null null 75 81 1.176 256 926 1.146 .988 62 80 439 457 0 3446 381 3066 1.359 .736 .442
17 3081 1444 427 336 2654 1108 null null 74 75 .422 256 929 .411 .981 62 80 463 481 0 1676 390 1286 .484 2.064 -1.046
18 845 587 392 323 453 264 null null 71 64 .584 256 748 .564 .844 89 107 80 98 0 681 375 306 .676 1.479 -.564
19 1269 955 394 319 875 636 null null 74 75 .729 256 721 .711 .957 89 107 104 122 0 1108 370 738 .843 1.186 -.246
20 635 439 396 321 239 118 null null 56 38 .541 256 705 .438 .655 89 107 128 146 0 509 372 137 .573 1.746 -.804

Total number of rows: 4608

Table truncated, full table size 529 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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