|
| Status |
Public on Dec 30, 2006 |
| Title |
X. fastidiosa in 3G10-R and Periwinkle Wilt media PW-Cy3_x_3G10R-Cy5_Exp2_Repl_2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Xylella fastidiosa cells grown in PW medium
|
| Organism |
Xylella fastidiosa 9a5c |
| Characteristics |
Total RNA from Xylella fastidiosa (isolate 9a5c), grown in PW medium until late exponential growth phase
|
| Biomaterial provider |
Luiz R. Nunes
|
| Treatment protocol |
N/A
|
| Growth protocol |
Cells of Xf 9a5c have been routinely kept in our laboratory in 20 ml of liquid cultures, which were incubated in an orbital shaker at 28oC and 100 rpm. For the microarray hybridization experiments, 1 ml aliquots of exponentially-growing cultures were transferred to 19 ml of fresh PW medium and incubated as mentioned above for 3 days, until an OD600 = 0.25 (late phase of exponential growth) was reached. This culture was then transferred to 300 ml of fresh medium and incubated for another 3 days, untill an OD600 = 0.25 was reached again. Cells were then harvested for RNA extraction
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from Xylella fastidiosa cells was extracted with the aid of the RNAeasy system (Qiagen), according to the manufacturer´s instructions
|
| Label |
Cy3-dCTP
|
| Label protocol |
Thirty micrograms (30 ug) of total P. brasiliensis RNA were mixed with 1 ul of a 4 ug/ul random primer solution (9-mers) in 20 ul of water and heated to 65oC for 10 min. The tube was briefly chilled on ice and mixed with 4 ul of 5mM dNTPs, 4 ul of 0.1 M DTT, 8 ul of 5X first-strand buffer (Invitrogen) and 1.5 ul of Superscript II (200 units/ml) (Invitrogen). The reaction was then incubated at 42oC for 1.5 h and terminated by a 15-min incubation at 65oC. The RNA was degraded by adding 1 ul of 10 mg/ml RNAse A and incubation at 37oC for 1 hour. The resulting first strand cDNA was purified and concentrated with the aid of a Microcon YM-30 cartridge and final volume was adjusted to 29 ul. One microliter (1 ul) of 4 ug/ul random primer solution (9-mers) and 4 ul of 10X Klenow buffer were then added to the system. The tube was then heated at 65oC for 5 min and allowed to cool to room temperature for ten min for primer annealing. Finally, fluorescence incorporation was performed by the addition of 4 ul of 10X low dNTP solution (5mM dATP, 5mM dTTP, 5mM dGTP and 2mM dCTP), 1 ul of Cy3-labeled dCTP (1mM) (Amersham Biosciences) and 1 ul of the Klenow fragment (50 units/ul) (Invitrogen). The reaction was then incubated at 37oC for 2 h and the final labeled cDNA was purified in a Microcon YM-30 cartridge (3 washes)
|
| |
|
| Channel 2 |
| Source name |
Xylella fastidiosa cells grown in 3G10R medium
|
| Organism |
Xylella fastidiosa 9a5c |
| Characteristics |
Total RNA from Xylella fastidiosa (isolate 9a5c), grown in 3G10R medium
|
| Biomaterial provider |
Luiz R. Nunes
|
| Treatment protocol |
N/A
|
| Growth protocol |
Cells of Xf 9a5c have been routinely kept in our laboratory in 20 ml of liquid cultures, which were incubated in an orbital shaker at 28oC and 100 rpm. For the microarray hybridization experiments, 1 ml aliquots of exponentially-growing cultures were transferred to 19 ml of fresh 3G10R medium and incubated as mentioned above for 13 days, until an OD600 = 0.25 was reached. This culture was then transferred to 300 ml of fresh medium and incubated for another 13 days, untill an OD600 = 0.25 was reached again. Cells were then harvested for RNA extraction
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from Xylella fastidiosa cells was extracted with the aid of the RNAeasy system (Qiagen), according to the manufacturer´s instructions
|
| Label |
Cy5-dCTP
|
| Label protocol |
Thirty micrograms (30 ug) of total P. brasiliensis RNA were mixed with 1 ul of a 4 ug/ul random primer solution (9-mers) in 20 ul of water and heated to 65oC for 10 min. The tube was briefly chilled on ice and mixed with 4 ul of 5mM dNTPs, 4 ul of 0.1 M DTT, 8 ul of 5X first-strand buffer (Invitrogen) and 1.5 ul of Superscript II (200 units/ml) (Invitrogen). The reaction was then incubated at 42oC for 1.5 h and terminated by a 15-min incubation at 65oC. The RNA was degraded by adding 1 ul of 10 mg/ml RNAse A and incubation at 37oC for 1 hour. The resulting first strand cDNA was purified and concentrated with the aid of a Microcon YM-30 cartridge and final volume was adjusted to 29 ul. One microliter (1 ul) of 4 ug/ul random primer solution (9-mers) and 4 ul of 10X Klenow buffer were then added to the system. The tube was then heated at 65oC for 5 min and allowed to cool to room temperature for ten min for primer annealing. Finally, fluorescence incorporation was performed by the addition of 4 ul of 10X low dNTP solution (5mM dATP, 5mM dTTP, 5mM dGTP and 2mM dCTP), 1 ul of Cy5-labeled dCTP (1mM) (Amersham Biosciences) and 1 ul of the Klenow fragment (50 units/ul) (Invitrogen). The reaction was then incubated at 37oC for 2 h and the final labeled cDNA was purified in a Microcon YM-30 cartridge (3 washes)
|
| |
|
| |
| Hybridization protocol |
Labelled cDNAs were mixed, dried in a Savant speed vacuum and resuspended in 100 ul of 1X Hybridization buffer, containing 6X SSC, 5X Denhardts solution, 0,25 mg/ml salmon sperm DNA, 0,01% SDS´and 50% formamide. Arrays were hybridized overnight (42oC) in a Gene-Tac Hybridization Station (Genomic Solutions, Inc., Ann Arbor, MI) and washed twice (42oC) in 0.5 X SSC, 0.01 % SDS, followed by two washes in 0.06 X SSC, 0.01 % SDS and two final washes in 0.06 X SSC. All washing steps consisted of 1 min of flow, followed by 5 min of incubation. Slides were then dried and subjected to fluorescent detection
|
| Scan protocol |
Slides were subjected to fluorescent detection with a GMS (Affymetrix) 418 Array Scanner (Affymetrix Inc., Santa Clara, CA) Cy3 channel was scanned at 100% laser power and 80% PMT gain Cy5 channel was scanned at 100% laser power and 80% PMT gain
|
| Description |
This hybridization is part of an experiment aimed at evaluating gene expression modulation during Xylella fastidiosa growth in two different media: PW (the standard complex medium normally used to cultivate X. fastidiosa under laboratory conditions) and 3G10R (a defined medium based on xylem chemical composition)
|
| Data processing |
Images were analyzed with the TIGR Spotfinder program (v.2.2.4). All spots with less than 50% of the pixels with values lower than the median local background plus two SD have been flagged and excluded from further analyses. Raw data were submitted to a series of mathematical transformations with the aid of the software TIGR MIDAS v.2.19. These included filtering out all spots whose integrated intensities were below 10,000 a/d units, normalization between the two channels with the aid of the Lowess algorithm and SD regularization of the Cy5/Cy3 ratios across all sectors (blocks) of the array
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| |
|
| Submission date |
Dec 27, 2006 |
| Last update date |
Dec 29, 2006 |
| Contact name |
Luiz R. Nunes |
| E-mail(s) |
luiz.nunes@ufabc.edu.br
|
| Phone |
(55) (11) 4798-7104
|
| Organization name |
UFABC - Universidade Federal do ABC
|
| Department |
Centro de Ciências Naturais e Humanas
|
| Lab |
Laboratório de Genômica Funcional e Estrutural
|
| Street address |
Rua Santa Adélia 166
|
| City |
Santo André |
| State/province |
SP |
| ZIP/Postal code |
09210-170 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL4683 |
| Series (1) |
| GSE6619 |
Transcriptome analysis of the phytobacterium Xylella fastidiosa growing under xylem-based chemical conditions |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
Normalized Log2(IB/IA) value |
| IA |
Raw integrated intensity value detected in channel A |
| IB |
Raw integrated intensity value detected in channel B |
| FlagA |
TIGR Spotfinder flag value in channel A |
| FlagB |
TIGR Spotfinder flag value in channel B |
| SA |
Actual spot area (in pixels) |
| SF |
Saturation factor |
| QCscore |
Cumulative quality control score |
| QCA |
Quality control score in channel A |
| QCB |
Quality control score in channel B |
| BkgA |
TIGR Spotfinder flag value in channel A |
| BkgB |
TIGR Spotfinder flag value in channel B |
