NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM886356 Query DataSets for GSM886356
Status Public on Mar 07, 2012
Title MTB1254 Day 0 rep 1
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 0
Organism Mycobacterium tuberculosis
Characteristics treatment: Control (Growth curve) 0 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/2007-06/75090.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 1151 277 115 1441 1036 null null 65 72 .747 293 1253 .695 .96 50 69 60 79 0 1266 127 1140 .791 1.264 -.338
2 750 459 277 113 473 346 null null 63 70 .721 293 1124 .684 .913 50 69 86 105 0 505 124 381 .805 1.243 -.313
3 2061 1550 286 115 1775 1435 null null 66 73 .818 293 1091 .771 .956 49 68 110 129 0 1705 127 1579 .889 1.124 -.169
4 790 569 303 128 487 441 null null 61 67 .864 293 1121 .862 .91 49 68 132 151 0 626 141 485 .996 1.004 -.006
5 3864 3564 295 125 3569 3439 null null 67 76 .984 293 1118 .931 .968 49 68 156 175 0 3921 138 3783 1.06 .943 .084
6 1485 1423 294 124 1191 1299 null null 65 72 1.115 293 1110 1.048 .956 49 68 180 199 0 1565 136 1429 1.2 .833 .263
7 694 444 298 124 396 320 null null 63 68 .808 293 1110 .767 .913 49 68 204 223 0 488 136 352 .889 1.125 -.17
8 1496 1299 288 121 1208 1178 null null 65 73 .946 293 1144 .914 .94 49 68 228 247 0 1429 133 1296 1.073 .932 .101
9 826 572 284 116 542 456 null null 63 67 .824 293 1142 .795 .935 49 68 254 273 0 629 128 502 .926 1.08 -.112
10 648 397 287 120 361 277 null null 61 68 .778 293 1108 .721 .899 49 68 278 297 0 437 132 305 .844 1.185 -.244
11 818 520 299 131 519 389 null null 64 69 .74 293 1100 .716 .905 49 68 302 321 0 572 144 428 .825 1.213 -.278
12 696 412 298 128 398 284 null null 64 67 .735 293 1111 .69 .903 48 67 326 345 0 453 141 312 .785 1.274 -.349
13 1451 1484 288 125 1163 1359 null null 62 69 1.152 293 1108 1.083 .928 48 67 350 369 0 1633 138 1495 1.285 .778 .362
14 722 533 289 132 433 401 null null 61 67 .93 293 1108 .877 .923 48 67 374 393 0 586 145 441 1.019 .982 .027
15 2182 1957 278 124 1904 1833 null null 63 71 .982 293 1120 .915 .952 48 67 398 417 0 2153 136 2016 1.059 .944 .083
16 3305 2844 263 114 3042 2730 null null 69 73 .918 293 1108 .836 .943 48 67 422 441 0 3129 125 3003 .987 1.013 -.018
17 2706 2390 267 115 2439 2275 null null 65 76 .934 293 1209 .88 .937 48 67 446 465 0 2629 127 2503 1.026 .975 .037
18 1296 936 284 114 1012 822 null null 62 70 .815 293 1110 .769 .952 74 93 62 81 0 1030 125 904 .894 1.119 -.162
19 1337 979 280 113 1057 866 null null 65 71 .814 293 973 .78 .932 74 93 86 105 0 1077 124 953 .901 1.11 -.15
20 354 157 280 113 74 44 null null 27 37 .63 293 963 .463 .587 73 92 108 127 0 173 124 48 .654 1.529 -.612

Total number of rows: 4608

Table truncated, full table size 528 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap