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Sample GSM137244 Query DataSets for GSM137244
Status Public on Sep 01, 2007
Title Low Iron 0vs060_L
Sample type RNA
 
Channel 1
Source name 28oC
Organism Xylella fastidiosa
Characteristics 28oC
Treatment protocol No treatment. Same as "Growth Protocol".
Growth protocol Xylella fastidiosa strain 9a5c isolated form CVC symptomatic Citrus (L.) Osbeck trees were grown in Periwinkle wilt (PW) broth media at 28oC with 80 rpm rotatory agitation. A culture started from a single colony was weekly passaged through serial transfers at a 1/10 dilution in PW medium.
Extracted molecule total RNA
Extraction protocol Total RNA was isolated with TRIzol (Invitrogen) and residual DNA was removed by treatment with of RQ1 RNAse-free DNAse I (Promega) and RNAse inhibitor RNAseOUT (Invitrogen). The integrity of the RNA was checked by agarose gel electrophoresis, quantization performed with a NanoDrop spectrophotometer and the lack of residual DNA was checked by PCR.
Label Alexa Fluor 555
Label protocol 20ug of total RNA was reverse transcribed and labeled using SuperScript Indirect cDNA labeling kit (Invitrogen). Unincorporated dye was removed by purification in Millipore filters MAFB000 in guanidine buffer pH 4.8 followed by 4 washes with ethanol 80%. Labeled cDNA was eluted with 90 ul of Tris 10mM, pH8.0 and dried in speed vac.
 
Channel 2
Source name low iron concentrarion, 60'
Organism Xylella fastidiosa
Characteristics low iron concentrarion, 60'
Treatment protocol Treated with 200 uM of 2,2-dipyridyl for 60 minutes.
Growth protocol Xylella fastidiosa strain 9a5c isolated form CVC symptomatic Citrus (L.) Osbeck trees were grown in Periwinkle wilt (PW) broth media at 28oC with 80 rpm rotatory agitation. A culture started from a single colony was weekly passaged through serial transfers at a 1/10 dilution in PW medium.
Extracted molecule total RNA
Extraction protocol Total RNA was isolated with TRIzol (Invitrogen) and residual DNA was removed by treatment with of RQ1 RNAse-free DNAse I (Promega) and RNAse inhibitor RNAseOUT (Invitrogen). The integrity of the RNA was checked by agarose gel electrophoresis, quantization performed with a NanoDrop spectrophotometer and the lack of residual DNA was checked by PCR.
Label Alexa Fluor 647
Label protocol 20ug of total RNA was reverse transcribed and labeled using SuperScript Indirect cDNA labeling kit (Invitrogen). Unincorporated dye was removed by purification in Millipore filters MAFB000 in guanidine buffer pH 4.8 followed by 4 washes with ethanol 80%. Labeled cDNA was eluted with 90 ul of Tris 10mM, pH8.0 and dried in speed vac.
 
 
Hybridization protocol AF555 and AF647 fluorescent labeled cDNAs were ressuspended in 13ul of water and mixed in a solution containing 25% of Hybridization Buffer (Amersham Biosciences) and 50% formamide, in a final volume of 56ul. Samples were denatured by heating at 95oC for 2 min and immediately chilled on ice. The targets were applied on the microarray slides, covered with a 24 x 60 mm coverslip (Corning) and hybridized at 42oC for 16h. Washing was performed once in 1xSSC, 0.1% SDS for 10 min, twice in 0.1xSSC, 0.01% SDS for 10 min and in 0.1xSSC for 1 min. The slides were soaked in water for 10s and dried with nitrogen gas.
Scan protocol Microarray slides were scanned with a Generation III DNA Scanner (Amersham Biosciences).
Description Fluorescence mean intensity and surrounding median background from each spot were obtained with ArrayVision v6.0 (Imaging Research, Inc). Unreliable spots, having intensities too similar to local background or saturated were filtered out. Spots presenting mean intensity below 2 times the standard deviation of its background in AF555 and AF647 simultaneously were eliminated from the subsequent analysis since their expression ratio is meaningless. Saturated signals [intensity greater than >990 fluorescence units] were also discarded. Normalization was carried out by LOWESS fitting on a M versus S plot, where M is the fluorescence log-ratio of the iron stress time-point relative to the control condition [M=log2(ratio)] and S is the log-mean fluorescence intensity [S = log2((cy3+cy5)/2)].
Data processing filtering low intensity spots, LOWESS normalization
 
Submission date Sep 21, 2006
Last update date Sep 22, 2006
Contact name Ricardo Z.N. Vêncio
Organization name Universidade de São Paulo
Department Computing and Mathematics Department
Lab http://labpib.fmrp.usp.br
Street address Av. Bandeirantes, 3900
City Ribeirao Preto
State/province SP
ZIP/Postal code 14049-900
Country Brazil
 
Platform ID GPL2708
Series (1)
GSE5888 Global gene expression analysis of the phytopathogen Xylella fastidiosa at low iron availability

Data table header descriptions
ID_REF
VALUE log2(normalized ratio)
C1.MTM Dens - RFU / ?m2 C1. Median-based Trimmed Mean density value for each spot. The reported value represents the mean of all the pixels remaining in a target, after first removing pixels with density values that exceed four median absolute deviations (MADs) above or below the median. This measure removes the influence of image artifacts (e.g., dust particles) on density estimation.
C1.% Removed (MTM - RFU/A) C1. Percentage of pixels excluded in the calculation of ARM Density and MTM Density.
C1.MAD - RFU / ?m2 C1. Median Absolute Deviation of foreground density.
C1.SD - RFU / ?m2 C1. Standard deviation of the pixel density values.
C1.Pos X - ?m x
C1.Pos Y - ?m y
C1.Area - ?m2 area
C1.Bkgd C1. Median of a region outside of the spot
C1.sMTMDens C1. Subtracted MTM Density value. MTM Density value of the spot, minus the background Density value.
C1.S/N C1. Signal-to-Noise Ratio. Spot density minus Background density, divided by the SD of the Background density.
C1.Flag Flag = 0 = O.K. ; other = bad spot
C1.% At Floor C1. Proportion of pixels at the lower limit of the density scale. Actual signal intensity may be below the imaging device's threshold of detection.
C1.% At Ceiling C1. Proportion of pixels at the upper limit of the density scale (i.e., saturated). Actual signal intensity may be higher than the recorded value.
C1.% At Floor - Bkgd C1. Backgr. Proportion of pixels at the lower limit of the density scale. Actual signal intensity may be below the imaging device's threshold of detection.
C1.% At Ceiling - Bkgd C1. Backgr. Proportion of pixels at the upper limit of the density scale (i.e., saturated). Actual signal intensity may be higher than the recorded value.
C2.MTM Dens - RFU / ?m2 C2. Median-based Trimmed Mean density value for each spot. The reported value represents the mean of all the pixels remaining in a target, after first removing pixels with density values that exceed four median absolute deviations (MADs) above or below the median. This measure removes the influence of image artifacts (e.g., dust particles) on density estimation.
C2.% Removed (MTM - RFU/A) C2. Percentage of pixels excluded in the calculation of ARM Density and MTM Density.
C2.MAD - RFU / ?m2 C2. Median Absolute Deviation of foreground density.
C2.SD - RFU / ?m2 C2. Standard deviation of the pixel density values.
C2.Pos X - ?m x
C2.Pos Y - ?m y
C2.Area - ?m2 area
C2.% At Floor C2. Proportion of pixels at the lower limit of the density scale. Actual signal intensity may be below the imaging device's threshold of detection.
C2.% At Ceiling C2. Proportion of pixels at the upper limit of the density scale (i.e., saturated). Actual signal intensity may be higher than the recorded value.
C2.Bkgd C2. Median of a region outside of the spot
C2.sMTMDens C2. Subtracted MTM Density value. MTM Density value of the spot, minus the background Density value.
C2.S/N C2. Signal-to-Noise Ratio. Spot density minus Background density, divided by the SD of the Background density.
C2.Flag Flag = 0 = O.K. ; other = bad spot
C2.% At Floor - Bkgd C2. Backgr. Proportion of pixels at the lower limit of the density scale. Actual signal intensity may be below the imaging device's threshold of detection.
C2.% At Ceiling - Bkgd C2. Backgr. Proportion of pixels at the upper limit of the density scale (i.e., saturated). Actual signal intensity may be higher than the recorded value.

Data table
ID_REF VALUE C1.MTM Dens - RFU / ?m2 C1.% Removed (MTM - RFU/A) C1.MAD - RFU / ?m2 C1.SD - RFU / ?m2 C1.Pos X - ?m C1.Pos Y - ?m C1.Area - ?m2 C1.Bkgd C1.sMTMDens C1.S/N C1.Flag C1.% At Floor C1.% At Ceiling C1.% At Floor - Bkgd C1.% At Ceiling - Bkgd C2.MTM Dens - RFU / ?m2 C2.% Removed (MTM - RFU/A) C2.MAD - RFU / ?m2 C2.SD - RFU / ?m2 C2.Pos X - ?m C2.Pos Y - ?m C2.Area - ?m2 C2.% At Floor C2.% At Ceiling C2.Bkgd C2.sMTMDens C2.S/N C2.Flag C2.% At Floor - Bkgd C2.% At Ceiling - Bkgd
1 0 91.78 1.563 7.282 10.151 685 58965 25600 97.881 0 0 0 0 0 0 0 9.077 1.953 0.824 1.133 695 58955 25600 0 0 9.734 0 0 0 0 0
2 0.38 108.258 7.422 5.118 8.311 945 58975 25600 96.089 12.169 2 0 0 0 0 0 11.793 1.172 1.152 1.522 955 58965 25600 0 0 9.615 2.178 1.651 0 0 0
3 4.43 94.276 1.563 4.172 5.563 1215 58995 25600 93.409 0.867 0.17 0 0 0 0 0 12.8 1.563 1.792 2.317 1225 58985 25600 0 0 9.325 3.475 2.765 0 0 0
4 0 84.238 0 5.589 7.301 1515 58965 25600 90.028 0 0 0 0 0 0 0 11.098 5.078 0.892 1.345 1525 58955 25600 0 0 9.216 1.881 1.738 0 0 0
5 0 73.212 1.172 5.86 7.915 1795 58965 25600 86.085 0 0 0 0 0 0 0 7.836 6.641 0.764 1.197 1805 58955 25600 0 0 9.162 0 0 0 0 0
6 0 85.371 3.125 4.788 6.577 2075 58965 25600 89.605 0 0 0 0 0 0 0 8.874 4.688 0.847 1.236 2085 58955 25600 0 0 9.771 0 0 0 0 0
7 0 87.461 1.563 6.097 8.265 2355 58965 25600 104.35 0 0 0 0 0 0 0 8.926 5.469 0.862 1.369 2365 58955 25600 0 0 10.731 0 0 0 0 0
8 0 102.374 7.422 3.788 6.118 2645 58965 25600 106.784 0 0 0 0 0 0 0 10.269 5.078 0.73 1.108 2655 58955 25600 0 0 10.905 0 0 0 0 0
9 0 96.872 5.469 5.343 9.468 2935 58965 25600 101.065 0 0 0 0 0 0 0 9.081 4.688 0.929 1.342 2945 58955 25600 0 0 10.403 0 0 0 0 0
10 0 93.766 1.953 3.161 4.1 3225 58965 25600 95.102 0 0 0 0 0 0 0 9.726 1.172 0.785 1.031 3235 58955 25600 0 0 9.822 0 0 0 0 0
11 0 82.973 0 5.742 7.241 3505 58965 25600 88.233 0 0 0 0 0 0 0 8.574 2.734 0.909 1.245 3515 58955 25600 0 0 9.754 0 0 0 0 0
12 -0.35 135.865 0.391 14.515 19.664 3785 58985 25600 83.813 52.052 11.972 0 0 0 0 0 14.503 2.344 1.613 2.23 3795 58975 25600 0 0 9.662 4.842 4.436 0 0 0
13 -0.28 278.468 0 98.091 106.085 4075 58985 25600 84.147 194.322 42.489 0 0 0 0 0 30.04 0 9.077 9.528 4085 58975 25600 0 0 9.659 20.381 18.352 0 0 0
14 -0.77 117.728 7.813 10.919 16.73 4365 58985 25600 84.607 33.121 7.49 0 0 0 0 0 12.509 7.813 1.191 1.857 4375 58975 25600 0 0 10.01 2.499 2.086 0 0 0
15 2.58 83.219 2.734 4.58 6.409 4655 58965 25600 82.96 0.259 0.061 0 0 0 0 0 10.284 5.469 0.741 1.1 4665 58955 25600 0 0 9.634 0.651 0.505 0 0 0
16 0 90.979 2.734 7.146 10.421 4945 58965 25600 82 8.979 2.054 0 0 0 0 0 7.242 0.391 1.073 1.391 4955 58955 25600 0 0 9.137 0 0 0 0 0
17 -0.96 92.913 5.469 3.866 5.918 5245 58985 25600 80.934 11.979 2.659 0 0 0 0 0 10.505 5.078 0.93 1.391 5255 58975 25600 0 0 9.593 0.912 0.202 0 0 0
18 -1.39 108.666 5.859 8.474 13.288 5535 58975 25600 81.956 26.709 1.581 0 0 0 0 0 11.287 3.125 0.957 1.35 5545 58965 25600 0 0 10.021 1.265 0.287 0 0 0
19 -0.98 168.313 7.422 22.099 34.565 5825 58975 25600 86.442 81.871 5.229 0 0 0 0 0 15.062 4.297 1.465 2.198 5835 58965 25600 0 0 9.681 5.381 4.741 0 0 0
20 0.45 161.454 3.125 19.224 28.174 6105 58975 25600 85.805 75.649 8.805 0 0 0 0 0 22.768 1.172 3.304 4.514 6115 58965 25600 0 0 9.53 13.238 12.526 0 0 0

Total number of rows: 4608

Table truncated, full table size 663 Kbytes.




Supplementary data files not provided
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