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Sample GSM886253 Query DataSets for GSM886253
Status Public on Mar 07, 2012
Title MTB strain 1254 Ctrl vs 0.05 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: 0.05 mM DETA/NO for 40min
Organism Mycobacterium tuberculosis
Characteristics strain name: 1254 (wild_type)
treatment: 0.05mM 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/2001-07/18440.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 7981 7577 1815 1498 6166 6079 1916 1678 95 91 .941 137 1429 .817 .793 109 122 164 177 0 9714 1921 7793 1.264 .791 .338
2 5046 3951 1724 1388 3322 2563 1803 1502 96 84 .678 137 1390 .646 .726 108 121 186 199 0 5065 1779 3286 .989 1.011 -.016
3 3723 3112 1716 1346 2007 1766 1789 1444 53 62 .931 137 1411 .611 .705 108 121 208 221 0 3990 1726 2264 1.128 .886 .174
4 2990 2310 1678 1367 1312 943 1758 1457 36 37 .934 137 1403 .45 .581 108 121 231 244 0 2962 1753 1209 .922 1.085 -.118
5 2640 2212 1654 1427 986 785 1737 1495 26 26 .86 137 1376 .497 .588 108 121 253 266 0 2836 1829 1007 1.021 .979 .03
6 2744 2327 1755 1467 989 860 1846 1540 29 33 .983 137 1403 .408 .594 108 121 276 289 0 2983 1881 1102 1.114 .897 .156
7 2983 2644 1790 1490 1193 1154 1881 1581 28 34 .825 137 1402 .509 .64 108 121 298 311 0 3390 1910 1479 1.24 .806 .31
8 3116 2678 1843 1528 1273 1150 1923 1622 31 37 .889 137 1369 .48 .614 108 121 321 334 0 3433 1959 1474 1.158 .864 .211
9 2783 2279 1845 1583 938 696 1904 1645 24 23 .947 137 1354 .462 .631 108 121 343 356 0 2921 2029 892 .951 1.052 -.073
10 27681 29124 1845 1590 25836 27534 1905 1686 82 80 1.025 177 1310 .896 .857 107 122 365 380 0 37339 2038 35301 1.366 .732 .45
11 18590 21991 1861 1552 16729 20439 1992 1868 63 67 1.229 177 1345 1.015 .869 105 120 387 402 0 28193 1990 26204 1.566 .638 .647
12 14708 14684 1722 1363 12986 13321 1877 1677 67 68 1.027 177 1358 .941 .898 105 120 410 425 0 18825 1747 17078 1.315 .76 .395
13 1782 1522 1589 1368 193 154 1661 1439 20 23 .772 137 1354 .411 .531 108 121 433 446 0 1952 1754 198 1.026 .975 .037
14 2792 2427 1742 1532 1050 895 1791 1586 28 26 .821 137 1380 .522 .639 108 121 456 469 0 3111 1964 1147 1.092 .916 .127
15 2984 2613 1808 1611 1176 1002 1882 1674 28 30 .91 137 1408 .593 .669 108 121 478 491 0 3350 2065 1285 1.093 .915 .128
16 3592 2696 1820 1605 1772 1091 1909 1666 33 29 .772 137 1397 .502 .672 108 121 501 514 0 3456 2058 1399 .789 1.267 -.341
17 4019 4353 1885 1648 2134 2705 1948 1724 56 75 1.131 137 1400 .754 .718 108 121 523 536 0 5581 2113 3468 1.625 .615 .701
18 7289 7786 2055 1720 5234 6066 2167 2024 88 96 1.263 137 1406 .862 .713 108 121 546 559 0 9982 2205 7777 1.486 .673 .571
19 10624 11163 2131 1639 8493 9524 2420 2022 96 100 1.06 137 1432 .892 .799 108 121 568 581 0 14312 2101 12211 1.438 .696 .524
20 2527 2322 1814 1621 713 701 1903 1735 28 31 1.119 137 1347 .425 .428 131 144 164 177 0 2977 2078 899 1.261 .793 .335

Total number of rows: 5760

Table truncated, full table size 725 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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