|
| Status |
Public on Dec 21, 2010 |
| Title |
J1a12 in XDM2 vs J1a12 in XDM0 for 8h - 3000137052L |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
J1a12 in XDM0 for 8h
|
| Organism |
Xylella fastidiosa |
| Characteristics |
strain: J1a12
treatment: XDM0
time: 8h
|
| Treatment protocol |
Wild type strain cells were grown during 12 days in the XDM2 medium (mid-log phase) and harvested by centrifugation. Cells were washed with XDM2 medium and cultivated in a medium lacking all nitrogen sources (XDM0). The cultivation was continued for 8 h.
|
| Growth protocol |
The citrus strain J1a12 of Xylella fastidiosa (Monteiro et al., 2001) was cultivated in PW medium (Davis et al., 1981) without bovine serum albumin and phenol red and supplemented with 0.5% glucose (w/v) (PWG) at 25°C with no agitation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from X. fastidiosa wild type and rpoN mutant cells, grown under nitrogen excess or nitrogen starvation conditions as described above, using the TRIZOL reagent (Invitrogen), according to the manufacturer's instructions. DNA was removed using RQ1 DNase I (Promega). RNA samples were evaluated by electrophoresis on formaldehyde-agarose gels and stored at -80°C.
|
| Label |
Cy3
|
| Label protocol |
20ug of total RNA was reverse transcribed and labeled using the CyScribe Post-labelling kit (GE Healthcare). 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 a speed vac.
|
| |
|
| Channel 2 |
| Source name |
J1a12 in XDM2
|
| Organism |
Xylella fastidiosa |
| Characteristics |
strain: J1a12
treatment: XDM2
|
| Treatment protocol |
Wild type strain cells were grown during 12 days in the XDM2 medium (mid-log phase) and harvested by centrifugation. Cells were washed with XDM2 medium.
|
| Growth protocol |
The citrus strain J1a12 of Xylella fastidiosa (Monteiro et al., 2001) was cultivated in PW medium (Davis et al., 1981) without bovine serum albumin and phenol red and supplemented with 0.5% glucose (w/v) (PWG) at 25°C with no agitation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from X. fastidiosa wild type and rpoN mutant cells, grown under nitrogen excess or nitrogen starvation conditions as described above, using the TRIZOL reagent (Invitrogen), according to the manufacturer's instructions. DNA was removed using RQ1 DNase I (Promega). RNA samples were evaluated by electrophoresis on formaldehyde-agarose gels and stored at -80°C.
|
| Label |
Cy5
|
| Label protocol |
20ug of total RNA was reverse transcribed and labeled using the CyScribe Post-labelling kit (GE Healthcare). 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 a speed vac.
|
| |
|
| |
| Hybridization protocol |
Cy3 and Cy5 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 shrinked? in water for 10s and dried with nitrogen gas.
|
| Scan protocol |
Microarray slides were scanned with a Generation III DNA Scanner (Amersham Biosciences).
|
| Description |
Gene expression analysis.
|
| Data processing |
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 Cy3 and Cy5 simultaneously were eliminated from the subsequent analysis since their expression ratio is meaningless. Saturated signals [intensity greater than >990 fluorescence units] were also discarded. R scripts (PMID 16755501) were used to normalize the data. Normalization was carried out by LOWESS fitting on a M versus S plot, where M is the fluorescence log-ratio of the test condition time-point relative to the control condition [M=log2(ratio)] and S is the log-mean fluorescence intensity [S = log2((Cy3+Cy5)/2)].
Filtering low intensity spots, LOWESS normalization.
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| |
|
| Submission date |
May 04, 2010 |
| Last update date |
Dec 21, 2010 |
| Contact name |
Tie Koide |
| E-mail(s) |
tiekoide@gmail.com
|
| Organization name |
Institute for Systems Biology
|
| Street address |
1441 N 34th street
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98103 |
| Country |
USA |
| |
|
| Platform ID |
GPL2708 |
| Series (1) |
| GSE21647 |
Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
log2 normalized (XDMO/XDM2) ratio |
| MTM Dens - RFU / µm2 |
Cy3. 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. |
| % Removed (MTM - RFU/A) |
Cy3. Percentage of pixels excluded in the calculation of ARM Density and MTM Density. |
| MAD - RFU / µm2 |
Cy3. Median Absolute Deviation of foreground density. |
| SD - RFU / µm2 |
Cy3. Standard deviation of the pixel density values. |
| Bkgd |
Cy3. Median of a region outside of the spot |
| sMTMDens |
Cy3. Subtracted MTM Density value. MTM Density value of the spot, minus the background Density value. |
| S/N |
Cy3. Signal-to-Noise Ratio. Spot density minus Background density, divided by the SD of the Background density |
| Flag |
0 = O.K. ; other = bad spot |
| % At Floor |
Cy3. Proportion of pixels at the lower limit of the density scale. Actual signal intensity may be below the imaging device's threshold of detection. |
| % At Ceiling |
Cy3. Proportion of pixels at the upper limit of the density scale (i.e., saturated). Actual signal intensity may be higher than the recorded value. |
| % At Floor - Bkgd |
Cy3. 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. |
| % At Ceiling - Bkgd |
Cy3. 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. |
| MTM Dens - RFU / µm2 |
Cy5. 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. |
| % Removed (MTM - RFU/A) |
Cy5. Percentage of pixels excluded in the calculation of ARM Density and MTM Density. |
| MAD - RFU / µm2 |
Cy5. Median Absolute Deviation of foreground density. |
| SD - RFU / µm2 |
Cy5. Standard deviation of the pixel density values. |
| % At Floor |
Cy5. Proportion of pixels at the lower limit of the density scale. Actual signal intensity may be below the imaging device's threshold of detection. |
| % At Ceiling |
Cy5. Proportion of pixels at the upper limit of the density scale (i.e., saturated). Actual signal intensity may be higher than the recorded value. |
| Bkgd |
Cy5. Median of a region outside of the spot |
| sMTMDens |
Cy5. Subtracted MTM Density value. MTM Density value of the spot, minus the background Density value. |
| S/N |
Cy5. Signal-to-Noise Ratio. Spot density minus Background density, divided by the SD of the Background density. |
| Flag |
0 = O.K. ; other = bad spot |
| % At Floor - Bkgd |
Cy5. 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. |
| % At Ceiling - Bkgd |
|
