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Sample GSM886255 Query DataSets for GSM886255
Status Public on Mar 07, 2012
Title MTB strain 1254 Ctrl vs 0.5 mM DETA/NO 40min rep 3
Sample type RNA
 
Channel 1
Source name Strain: MTB strain 1254 Treatment: Control
Organism Mycobacterium tuberculosis
Characteristics strain name: 1254 (wild_type)
treatment: control
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 Strain: MTB strain 1254 Treatment: 0.5 mM DETA/NO for 40min
Organism Mycobacterium tuberculosis
Characteristics strain name: 1254 (wild_type)
treatment: 0.5mM of diethylenetriamine/nitric oxide for 40 minutes
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: Stress, Stress by growth condition
Image: http://smd.stanford.edu/MicroArray/gifs/2007-06/74759.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 GPL8561
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 18984 13985 2291 1573 16693 12412 null null 73 70 .727 208 1319 .669 .888 91 107 175 191 0 18963 2133 16830 1.008 .992 .012
2 17197 13356 2492 1723 14705 11633 null null 72 69 .771 208 1251 .706 .876 91 107 197 213 0 18110 2336 15774 1.073 .932 .101
3 13129 9604 3192 1792 9937 7812 null null 66 65 .744 208 1251 .663 .835 91 107 220 236 0 13022 2430 10593 1.066 .938 .092
4 12243 9219 3598 1837 8645 7382 null null 58 62 .839 208 1251 .69 .861 91 107 242 258 0 12500 2491 10010 1.158 .864 .211
5 9677 6842 3503 1609 6174 5233 null null 56 56 .878 208 1251 .648 .792 91 107 265 281 0 9277 2182 7096 1.149 .87 .201
6 7762 5228 3194 1524 4568 3704 null null 53 59 .77 208 1292 .593 .773 91 107 287 303 0 7089 2066 5022 1.099 .91 .137
7 2124 1282 2431 1472 -307 -190 null null 6 3 .346 137 1361 .164 .506 93 106 312 325 0 1738 1996 -258 null null null
8 3989 2723 2251 1433 1738 1290 null null 48 52 .619 137 1381 .568 .754 93 106 334 347 0 3692 1943 1749 1.006 .994 .009
9 9575 6166 2179 1391 7396 4775 null null 76 73 .606 137 1381 .526 .795 93 106 357 370 0 8361 1886 6475 .875 1.142 -.192
10 5945 5474 2123 1361 3822 4113 null null 59 69 .802 137 1381 .826 .782 93 106 379 392 0 7422 1845 5577 1.459 .685 .545
11 1795 1177 2096 1368 -301 -191 null null 4 3 .397 137 1381 .163 .316 93 106 402 415 0 1596 1855 -259 null null null
12 2826 1964 2619 1797 207 167 null null 20 10 .453 137 1381 .279 .491 93 106 424 437 0 2663 2437 226 1.094 .914 .13
13 2870 1960 2957 2052 -87 -92 null null 7 3 .576 137 1381 .255 .427 93 106 447 460 0 2658 2782 -125 null null null
14 2799 1984 2514 1756 285 228 null null 19 15 .588 137 1381 .255 .44 93 106 469 482 0 2690 2381 309 1.085 .922 .117
15 1802 1297 2126 1468 -324 -171 null null 4 4 .842 137 1381 .244 .407 93 106 492 505 0 1759 1991 -232 null null null
16 2744 1971 2004 1394 740 577 null null 34 41 .62 137 1381 .289 .484 93 106 514 527 0 2673 1890 782 1.057 .946 .08
17 1901 1350 2139 1552 -238 -202 null null 7 7 .503 137 1381 .3 .453 93 106 537 550 0 1831 2104 -274 null null null
18 2177 1599 2264 1674 -87 -75 null null 9 9 .648 137 1381 .312 .476 93 106 559 572 0 2168 2270 -102 null null null
19 1969 1498 2404 1762 -435 -264 null null 1 0 .393 137 1447 .814 .841 93 106 582 595 0 2031 2389 -358 null null null
20 2816 2035 2877 1762 -61 273 null null 1 7 .803 137 1351 .32 .396 115 128 176 189 -100 2759 2389 370 null null null

Total number of rows: 5760

Table truncated, full table size 707 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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