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Sample GSM642813 Query DataSets for GSM642813
Status Public on Dec 01, 2011
Title Transcriptional profiling of Escherichia coli during a transition from zinc starvation to surfeit (slide 10A1)
Sample type RNA
 
Channel 1
Source name E. coli 10 mins
Organism Escherichia coli
Characteristics strain: MG1655
agent: H2O
time: 10 mins
Extracted molecule total RNA
Extraction protocol Zn-depleted custom-built chemostats were grown for 50 h. At this point, ZnSO4.7H2O in water was added to a final concentration of 0.2 M in the chemostat. A 10 ml sample of culture was taken using a polypropylene pipette tip immediately prior to Zn addition and 2.5, 7, 10 and 30 min after addition. The culture was pipetted directly into RNAprotect (Qiagen) to stabilize RNA. Total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers. RNA was quantified using a BioPhotometer (Eppendorf). A control experiment was carried out in which water was added.
Label Cy3
Label protocol RNA (15-20 micrograms) was annealed to 9 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC. This was supplemented with 4 microlitres of 5× First-strand buffer (Invitrogen), 2 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 2 microlitres l 0.1 M DTT, 2 microlitres 1 mM Cy3 (Perkin Elmer) and 1 microlitre Superscript II reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 2 hours. The reaction was terminated by the addition of NaOH (5 microlitres of 1 M) and HCl (5 microlitres of 1 M) before the addition of TE buffer (200 microlitres). Labelled cDNA was purified using a Qiaquick purification kit (Qiagen).
 
Channel 2
Source name E. coli 10 mins
Organism Escherichia coli
Characteristics strain: MG1655
agent: ZnSO4
time: 10 mins
Extracted molecule total RNA
Extraction protocol Zn-depleted custom-built chemostats were grown for 50 h. At this point, ZnSO4.7H2O in water was added to a final concentration of 0.2 M in the chemostat. A 10 ml sample of culture was taken using a polypropylene pipette tip immediately prior to Zn addition and 2.5, 7, 10 and 30 min after addition. The culture was pipetted directly into RNAprotect (Qiagen) to stabilize RNA. Total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers. RNA was quantified using a BioPhotometer (Eppendorf). A control experiment was carried out in which water was added.
Label Cy5
Label protocol RNA (15-20 micrograms) was annealed to 9 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC. This was supplemented with 4 microlitres of 5× First-strand buffer (Invitrogen), 2 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 2 microlitres l 0.1 M DTT, 2 microlitres 1 mM Cy5 (Perkin Elmer) and 1 microlitre Superscript II reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 2 hours. The reaction was terminated by the addition of NaOH (5 microlitres of 1 M) and HCl (5 microlitres of 1 M) before the addition of TE buffer (200 microlitres). Labelled cDNA was purified using a Qiaquick purification kit (Qiagen).
 
 
Hybridization protocol The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 °C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 °C) SSC buffers for 5 min each at 37oC: 1× SSC/0.1 % SDS, 1× SSC, 0.2× SSC and 0.01× SSC. Microarray slides were dried by centrifugation at 500 × g for 2 min before scanning.
Scan protocol Slides were scanned on an Affymetrix 428 scanner.
Data processing The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5).
Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values.
 
Submission date Dec 20, 2010
Last update date Dec 01, 2011
Contact name Robert Poole
E-mail(s) R.poole@shef.ac.uk
Phone 01142224447
Organization name University of Sheffield
Department Molecular Biology & Biotechnology
Lab F13
Street address Firth Court, Western Bank
City Sheffield
State/province South Yorkshire
ZIP/Postal code S10 2TN
Country United Kingdom
 
Platform ID GPL534
Series (1)
GSE26187 Transcriptional profiling of Escherichia coli during a transition from zinc starvation to surfeit

Data table header descriptions
ID_REF
VALUE -[INV_VALUE]; (ZnSO4/H2O) ratio following background correction, log2 transformation, Loess normalisation
CH1_MEAN Mean data in Cy3 Channel
CH1_BKD_MEAN Background Mean in Cy3 Channel
CH1_AREA Spot Area in Cy3 Channel
CH1_BKD_AREA Background Area in Cy3 Channel
CH1_RAW Raw signal output data obtained from image analysis algorithm in Cy3 Channel
CH1_BKD_RAW Raw background output data obtained from image analysis algorithm in Cy3 Channel
CH1_SD Standard Deviation obtained in Cy3 Channel
CH1_BKD_SD Background Standard Deviation in Cy3 Channel
CH2_MEAN Mean data in Cy5 Channel
CH2_BKD_MEAN Background Mean in Cy5 Channel
CH2_AREA Spot Area in Cy5 Channel
CH2_BKD_AREA Background Area in Cy5 Channel
CH2_RAW Raw signal output data obtained from image analysis algorithm in Cy5 Channel
CH2_BKD_RAW Raw background output data obtained from image analysis algorithm in Cy5 Channel
CH2_SD Standard Deviation obtained in Cy5 Channel
CH2_BKD_SD Background Standard Deviation in Cy5 Channel
INV_VALUE VALUE Cy3:Cy5 ratio following background correction, log2 transformation, Loess normalisation

Data table
ID_REF VALUE CH1_MEAN CH1_BKD_MEAN CH1_AREA CH1_BKD_AREA CH1_RAW CH1_BKD_RAW CH1_SD CH1_BKD_SD CH2_MEAN CH2_BKD_MEAN CH2_AREA CH2_BKD_AREA CH2_RAW CH2_BKD_RAW CH2_SD CH2_BKD_SD INV_VALUE
1.1.1.1 1541.53719 595.8569444 121 720 186526 429017 515.4290525 210.4895955 532.1162791 368.2436472 172 669 91524 246355 269.2833909 193.939627
1.1.1.2 0.0591506 1918.851852 623.3513932 54 646 103618 402685 527.2843216 232.0335503 684.4888889 378.4015267 45 655 30802 247853 255.7797336 213.6119811 -0.059150597
1.1.1.3 -0.282229 2833.177419 643.0539033 62 538 175657 345963 865.393711 290.5745924 1506.561404 362.012987 57 539 85874 195125 541.4175033 206.2156719 0.282228514
1.1.1.4 0.66471 2720.424658 579.7816291 73 577 198591 334534 768.2629565 244.7564364 855.6727273 355.0382609 55 575 47062 204147 390.1029726 209.6778281 -0.664710471
1.1.1.5 0.115945 2837.188406 646.7625899 69 556 195766 359600 715.3834337 243.9540463 1203.533333 391.5185841 60 565 72212 221208 380.3195352 220.6364288 -0.115945446
1.1.1.6 0.410772 12514.46575 614.4211438 73 577 913556 354521 5660.936715 320.4004769 7056.901408 361.1519862 71 579 501040 209107 3310.028042 203.5587299 -0.410771513
1.1.1.7 0.523668 11764.21348 928.2054795 89 511 1047015 474313 5502.868205 2521.646671 5974.647727 444.2265625 88 512 525769 227444 2965.005715 305.8505613 -0.523667665
1.1.1.8 1.29018 1310.3 554.4768612 80 497 104824 275575 401.539145 211.4989523 360.826087 379.5293006 69 529 24897 200771 175.4373381 272.4497345 -1.290177087
1.1.1.9 -0.264034 5272.591549 513.9529837 71 553 374354 284216 2232.958144 243.0925704 3945.742424 368.9265233 66 558 260419 205861 1543.480014 223.6803514 0.264033683
1.1.1.10 -0.0162476 22253.46392 503.5587703 97 553 2158586 278468 11753.29253 287.2697768 17971.89412 397.1823009 85 565 1527611 224408 7855.086811 351.7439138 0.016247583
1.1.1.11 0.444082 15584.91139 521.9089317 79 571 1231208 298010 7488.684653 339.0696173 8898.594595 349.6041667 74 576 658496 201372 3839.828994 243.4507271 -0.444081571
1.1.1.12 0.102184 2871.2 465.0470588 80 595 229696 276703 1037.055367 234.1777127 1264.298507 321.307309 67 602 84708 193427 421.8862411 187.4135909 -0.10218409
1.1.1.13 0.46514 1680.655738 461.770798 61 589 102520 271983 431.7390258 231.5171709 515.4444444 357.2715232 46 604 23195 215792 239.1802255 200.1630191 -0.465140308
1.1.1.14 0.0943948 11988.41121 466.7818533 107 518 1282760 241793 6679.789376 284.4790685 8557.340909 339.6182495 88 537 753046 182375 4057.323547 208.0545997 -0.0943948
1.1.1.15 0.344267 5188.707317 445.4130435 123 552 638211 245868 2446.274449 209.0444263 2458.509804 336.7434555 102 573 250768 192954 819.7462752 196.1295886 -0.344266727
1.1.1.16 0.10993 1972.229508 466.1675302 61 579 120306 269911 432.506359 240.5380941 766.5925926 318.2414414 54 555 41396 176624 226.542819 190.1397383 -0.109930375
1.1.1.17 -0.483002 2655.945946 355.0210526 74 570 196540 202362 1013.887046 182.446838 1757.375 288.2448276 64 580 112472 167182 616.8637284 171.2193251 0.483001588
1.1.1.18 1133.046154 365.1689189 65 592 73648 216180 317.4074029 176.7540048 347.15 296.7495908 60 611 20829 181314 188.2640721 180.709389
1.1.2.1 0.0107078 4488.888889 777.137931 180 522 808000 405666 1531.516572 322.8330057 2428.871951 484.6412639 164 538 398335 260737 780.6382407 262.5620754 -0.010707816
1.1.2.2 -0.066806 2232.626374 694.1910499 91 581 203169 403325 558.2699857 285.8255844 980.1758242 405.7765957 91 564 89196 228858 318.57718 227.3603767 0.06680595

Total number of rows: 4608

Table truncated, full table size 742 Kbytes.




Supplementary data files not provided
Processed data included within Sample table

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