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| Status |
Public on Mar 03, 2009 |
| Title |
P. syringae pv. phaseolicola NPS3121 responses to apoplastic fluid biological replicate 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
P. syringae pv. phaseolicola NPS3121 with apoplastic fluid
|
| Organism |
Pseudomonas savastanoi pv. phaseolicola |
| Characteristics |
strain: NPS3121
|
| Treatment protocol |
For microarray experiments, P. syringae pv. phaseolicola NPS3121 was inoculated and grown on M9 medium at 18 oC to an optical density of 0.6 (OD 600nm). The culture was then divided into two equal parts; to one culture apoplastic fluid of bean leaf was added, while the other culture was used without extract additions. Both cultures were grown under the same conditions. Test culture for 6 hours, and control culture to reach a optical density similar to control culture.
|
| Growth protocol |
Overnight culture of strain NPS3121 was used to inoculate the fresh minimal medium M9, and it was allowed to grow at 18 °C to the mid exponential growth phase (A600 = 0.6).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol reagent as recommended by the manufacturer (Invitrogen). A second step of purification was performed using RNeasy MinElute spin columns (Qiagen) to remove contaminating DNA. RNAs were eluted in 50 µl of diethylpyrocarbonate (DEPC)-treated water and the concentration was determined using the NanoDrop equipment. RNA integrity was checked by analytical agarose gel electrophoresis
|
| Label |
Cy5
|
| Label protocol |
Purified total RNA (30 ug) was mixed with 3 uL of random nonamer primers and was incubated at 70ºC for 5 min. Reactions were held at room temperature for 10 min to allow the primers and the RNA template to anneal. To each reaction, the following were added: 1 uL of Cy5/Cy3-dCTP (1 mM) (Amersham Biosciences, Piscataway, NJ, U.S.A.), 2 uL of 0.1 M dithiothreitol, 4 uL of 5X Cyscript buffer, 1 uL of dNTP mixture (15 mM dATP, dTTP, and dGTP; 5 mM dCTP), 1uL of Cy3/Cy5 labeled dUTP and 200 U of CyScript reverse transcriptase. Reactions were incubated at 42ºC for 2 h. CyDye labelled single strand cDNA using the CyScribe GFX Purification Kit (Amersham Biosciences, Piscataway, NJ, U.S.A.) and according to the protocol recommended by the supplier. The purified labeled cDNA was then quantified to have with a Nanodrop equipment to final amount of 150 pg of each labeled cDNA.
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| |
|
| Channel 2 |
| Source name |
Pseudomonas syringae pv. phaseolicola NPS3121 whitout apoplastic fluid
|
| Organism |
Pseudomonas savastanoi pv. phaseolicola |
| Characteristics |
strain: NPS3121
|
| Treatment protocol |
P. syringae pv. phaseolicola NPS3121 was inoculated and grown on M9 medium at 18o C to an optical density of 0.6 (OD 600nm). This culture are considered as positive control of experiment, this was not supplemented with any extract.
|
| Growth protocol |
Overnight culture of strain NPS3121 was used to inoculate the fresh minimal medium M9, and it was allowed to grow at 18 °C to the mid exponential growth phase (A600 = 0.6).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol reagent as recommended by the manufacturer (Invitrogen). A second step of purification was performed using RNeasy MinElute spin columns (Qiagen) to remove contaminating DNA. RNAs were eluted in 50 µl of diethylpyrocarbonate (DEPC)-treated water and the concentration was determined using the NanoDrop equipment. RNA integrity was checked by analytical agarose gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
Purified total RNA (30 ug) was mixed with 3 uL of random nonamer primers and was incubated at 70ºC for 5 min. Reactions were held at room temperature for 10 min to allow the primers and the RNA template to anneal. To each reaction, the following were added: 1 uL of Cy5/Cy3-dCTP (1 mM) (Amersham Biosciences, Piscataway, NJ, U.S.A.), 2 uL of 0.1 M dithiothreitol, 4 uL of 5X Cyscript buffer, 1 uL of dNTP mixture (15 mM dATP, dTTP, and dGTP; 5 mM dCTP), 1uL of Cy3/Cy5 labeled dUTP and 200 U of CyScript reverse transcriptase. Reactions were incubated at 42ºC for 2 h. CyDye labelled single strand cDNA using the CyScribe GFX Purification Kit (Amersham Biosciences, Piscataway, NJ, U.S.A.) and according to the protocol recommended by the supplier. The purified labeled cDNA was then quantified to have with a Nanodrop equipment to final amount of 150 pg of each labeled cDNA.
|
| |
|
| |
| Hybridization protocol |
Labeled probes dried and resuspended in 40 µl of hybridization solution (25% hybridization buffer supplied in the CyScribe kit, 25% nuclease free water and 50% formamide). The probe was immediately applied onto the array and covered with a cover slip (Hybri-slips, Sigma-Aldrich Co. St Louis U.S.A.), hybridized 16 hours at 45°C in a humid hybridization chamber (Corning Incorporated, N.Y., U.S.A).
After hybridization, the slides were washed once in solutions 1–4 (wash solution 1: 2% SSC, 0.1% SDS at 45 ºC; wash solution 2: 0.1% SSC, 0.1% SDS; wash solution 3: 0.1% SSC; wash solution 4: 0.01% SSC) for 5 min with gentle stirring/agitation. Washed slides were dried by centrifugation at 1600 r.p.m
|
| Scan protocol |
Slides were scanned with an Axon GenePix 4000 B scanner at a resolution of 10 µm adjusting the laser and gain parameters to obtain similar levels of fluorescence intensity in both channels
|
| Description |
P. syringae pv. phaseolicola NPS3121 were inoculated and grown on M9 medium at 18o C to an optical density of 0.6 (OD 600nm). The culture was then divided into two equal parts; to one culture apoplastic fluid of bean pod was added, while the other culture was used without extract additions. Both cultures were grown under the same conditions. Test culture for 6 hours, and control culture to reach a optical density similar to control culture.
|
| Data processing |
Spot intensities were quantified using Axon GenePix Pro 6.0 image analysis software. The mean of the signals and the median of backgrounds were used for further analysis. Raw data were imported into the R 2.2.1 software (http://www.R-project.org). Background correction was done using the method Robust Multichip Analysis "RMA" (Irizarry et al., 2003) whereas normalization of the signal intensities within slides was carried out using the "printtiploess" method (Yang et al., 2002) using the LIMMA package (Smyth et al., 2003, www.bioconductor.org). Normalized data were log2 transformed and then fitted into mixed model ANOVAs (Gibson and Wolfinger, 2004; Wolfinger et al., 2001) using the Mixed procedure. The p-values of the extract effects were adjusted for by the False Discovery Rate method “FDR” (Benjamini and Hochberg, 1995). Estimates of the expression differences were calculated using the mixed model. Based on these statistical analyses, the spots with tests with an FDR less or equal to 5% and with changes in signal intensity between cultures with extract and culture control without extract of 1.5 fold or higher difference were considered as differentially expressed.
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| Submission date |
Feb 24, 2009 |
| Last update date |
Feb 28, 2009 |
| Contact name |
Jose Luis Hernandez-Flores |
| E-mail(s) |
[email protected]
|
| Organization name |
Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional
|
| Department |
Ingeniería Genética
|
| Lab |
Biología Molecular de Bacterias I
|
| Street address |
Km 9.6 Libramiento Norte Carretera Irapuato-León
|
| City |
Irapuato |
| State/province |
Guanajuato |
| ZIP/Postal code |
36821 |
| Country |
Mexico |
| |
|
| Platform ID |
GPL7115 |
| Series (1) |
| GSE14983 |
P. syringae pv. phaseolicola NPS3121 responses to apoplastic fluid of bean leaf |
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