|
| Status |
Public on Jun 01, 2006 |
| Title |
40oC 07min vs 29oC - 3000030720R |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
40oC 07min (cy3)
|
| Organism |
Xylella fastidiosa |
| Characteristics |
40oC 07min (cy3)
|
| Treatment protocol |
Heat shock at 40oC was applied to 7 days old cultures, using a water bath incubator with rotatory agitation of 150 rpm. 50ml aliquots of cell cultures were taken at time point 07 minutes and immediately harvested for RNA extraction.
|
| Growth protocol |
Xylella fastidiosa strain 9a5c isolated form CVC symptomatic Citrus (L.) Osbeck trees were grown in Periwinkle wilt (PW) broth media at 29oC with 100 rpm rotatory agitation in the dark. 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 phenol-chloroform (Ausubel , 1995) and residual DNA was removed by treatment with 10U of RQ1 RNAse-free DNAse I (Promega) and 40U RNAse inhibitor RNAsin (Promega). The integrity of the RNA was checked by agarose gel electrophoresis and the lack of residual DNA was checked by PCR.
|
| Label |
cy3
|
| Label protocol |
20ug of total RNA was reverse transcribed and labeled using 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 speed vac.
|
| |
|
| Channel 2 |
| Source name |
29oC (cy5)
|
| Organism |
Xylella fastidiosa |
| Characteristics |
29oC (cy5)
|
| Growth protocol |
Xylella fastidiosa strain 9a5c isolated form CVC symptomatic Citrus (L.) Osbeck trees were grown in Periwinkle wilt (PW) broth media at 29oC with 100 rpm rotatory agitation in the dark. 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 phenol-chloroform (Ausubel , 1995) and residual DNA was removed by treatment with 10U of RQ1 RNAse-free DNAse I (Promega) and 40U RNAse inhibitor RNAsin (Promega). The integrity of the RNA was checked by agarose gel electrophoresis and the lack of residual DNA was checked by PCR.
|
| Label |
cy5
|
| Label protocol |
20ug of total RNA was reverse transcribed and labeled using 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 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 |
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. Normalization was carried out by LOWESS fitting on a M versus S plot, where M is the fluorescence log-ratio of the heat shock 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 |
Feb 02, 2006 |
| Last update date |
Jun 01, 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) |
| GSE4161 |
Global gene expression analysis of the heat shock response in the phytopathogen Xylella fastidiosa |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
log2(normalized ratio) |
| Cy3.MTM Dens - RFU / �m2 |
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. |
| Cy3.% Removed (MTM - RFU/A) |
Cy3. Percentage of pixels excluded in the calculation of ARM Density and MTM Density. |
| Cy3.MAD - RFU / �m2 |
Cy3. Median Absolute Deviation of foreground density. |
| Cy3.SD - RFU / �m2 |
Cy3. Standard deviation of the pixel density values. |
| Cy3.Pos X - �m |
x |
| Cy3.Pos Y - �m |
y |
| Cy3.Area - �m2 |
area |
| Cy3.Bkgd |
Cy3. Median of a region outside of the spot |
| Cy3.sMTMDens |
Cy3. Subtracted MTM Density value. MTM Density value of the spot, minus the background Density value. |
| Cy3.S/N |
Cy3. Signal-to-Noise Ratio. Spot density minus Background density, divided by the SD of the Background density. |
| Cy3.Flag |
0 = O.K. ; other = bad spot |
| Cy3.% 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. |
| Cy3.% 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. |
| Cy3.% 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. |
| Cy3.% 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. |
| Cy5.MTM Dens - RFU / �m2 |
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. |
| Cy5.% Removed (MTM - RFU/A) |
Cy5. Percentage of pixels excluded in the calculation of ARM Density and MTM Density. |
| Cy5.MAD - RFU / �m2 |
Cy5. Median Absolute Deviation of foreground density. |
| Cy5.SD - RFU / �m2 |
Cy5. Standard deviation of the pixel density values. |
| Cy5.Pos X - �m |
x |
| Cy5.Pos Y - �m |
y |
| Cy5.Area - �m2 |
area |
| Cy5.% 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. |
| Cy5.% 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. |
| Cy5.Bkgd |
Cy5. Median of a region outside of the spot |
| Cy5.sMTMDens |
Cy5. Subtracted MTM Density value. MTM Density value of the spot, minus the background Density value. |
| Cy5.S/N |
Cy5. Signal-to-Noise Ratio. Spot density minus Background density, divided by the SD of the Background density. |
| Cy5.Flag |
0 = O.K. ; other = bad spot |
| Cy5.% 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. |
