|
| Status |
Public on Nov 17, 2011 |
| Title |
E. coli K12 interacted with lettuce rhizosphere rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Escherichia coli MG1655 cells interacted with the lettuce rhizosphere for 3 days
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
treatment: exposed to the lettuce rhizosphere for 3 days
|
| Treatment protocol |
Following incubation, roots had been gently rinsed to remove loosely attached bacteria and roots were cut directly into 100 ml ice cold stop solution (5 % H2O-saturated phenol, pH 4.3, in 95% ethanol). Root extracts were filtered using 0.5 µm filters to separate bacteria cells from plant cells in order to prevent interference of the cDNA labeling. Qiagen RNA protect solution was used to stabilize RNA during extraction. Bacteria had been collected by shaking and washing and stored in -80°C until using for RNA isolation.
|
| Growth protocol |
Escherichia coli MG1655 were grown in 10 ml of LB medium until stationary phase, collected by centrifugation at 8,000 ×g for 10 min, and washed twice with sterilized, plant growth medium (Caspersen et al. 1999). The resultant cells were re-suspended in 10 ml of the same plant growth medium to a final concentration of approximately 10E9 cells/ml. Germinated lettuce seedlings were each aseptically transferred into holes in the growth boxes filled with 300 ml of plant growth medium, and 3 ml washed bacterial suspension was added to provide approximately 10E7 cells/ml of growth medium. Seedling boxes were germinated for a week and transferred to a plant growth chamber, and the aeration pump (Luft Pump; Oceanic systems Inc. Dallas, TX) was immediately started. Each pump supplied aeration of three growth boxes, at a rate of 1.3 L /min. The seedling boxes were incubated under 80% RH at 25°C with a photoperiod of 16 h for 3 days
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Standard hot-phenol method
|
| Label |
Cy3
|
| Label protocol |
The total nucleic acid samples was precipitated and treated with DNase I for 30 min to remove contaminating DNA. The concentration of RNA was measured spectrophotometrically and RNA samples were electrophoresed in 0.8% agarose gels prior to use to ensure RNA integrity. The cDNA was synthesized from 10 µg of total RNA template.
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| |
|
| Channel 2 |
| Source name |
Escherichia coli MG1655 cells grown in the hydroponic system without interacting with the lettuce rhizosphere for 3 days
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
treatment: control
|
| Treatment protocol |
Following incubation, roots had been gently rinsed to remove loosely attached bacteria and roots were cut directly into 100 ml ice cold stop solution (5 % H2O-saturated phenol, pH 4.3, in 95% ethanol). Root extracts were filtered using 0.5 µm filters to separate bacteria cells from plant cells in order to prevent interference of the cDNA labeling. Qiagen RNA protect solution was used to stabilize RNA during extraction. Bacteria had been collected by shaking and washing and stored in -80°C until using for RNA isolation.
|
| Growth protocol |
Escherichia coli MG1655 were grown in 10 ml of LB medium until stationary phase, collected by centrifugation at 8,000 ×g for 10 min, and washed twice with sterilized, plant growth medium (Caspersen et al. 1999). The resultant cells were re-suspended in 10 ml of the same plant growth medium to a final concentration of approximately 10E9 cells/ml. Germinated lettuce seedlings were each aseptically transferred into holes in the growth boxes filled with 300 ml of plant growth medium, and 3 ml washed bacterial suspension was added to provide approximately 10E7 cells/ml of growth medium. Seedling boxes were germinated for a week and transferred to a plant growth chamber, and the aeration pump (Luft Pump; Oceanic systems Inc. Dallas, TX) was immediately started. Each pump supplied aeration of three growth boxes, at a rate of 1.3 L /min. The seedling boxes were incubated under 80% RH at 25°C with a photoperiod of 16 h for 3 days
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Standard hot-phenol method
|
| Label |
Cy5
|
| Label protocol |
The total nucleic acid samples was precipitated and treated with DNase I for 30 min to remove contaminating DNA. The concentration of RNA was measured spectrophotometrically and RNA samples were electrophoresed in 0.8% agarose gels prior to use to ensure RNA integrity. The cDNA was synthesized from 10 µg of total RNA template.
|
| |
|
| |
| Hybridization protocol |
Total RNA was converted to cDNA using the Superscript II Reverse transcriptase and directly labeled with Cy3 or Cy5 dyes (invitrogen) prior to hybridization. The labeled cDNA was applied to microarray slides. Following prehybridization, microarrays were hybridized with labeled cDNAs for 48 hours at 42°C in a water bath under dark
|
| Scan protocol |
Image analysis was carried out with the corresponding software program using a file that lists the corresponding gene to each spot. Signal intensities for each spot on scanned image files were determined using GenePix® Pro 6.0 software
|
| Data processing |
Signal intensities were normalized for spot and slide abnormalities with the spatial Lowess algorithm and analyzed by mixed-effect ANOVA (MAANOVA) (Kerr et al. 2000). Both Lowess and MAANOVA are part of the R/MAANOVA microarray statistical package, available at (http://www.jax.org/staff/churchill/labsite/). The resulting variety-by-gene interaction (VG) values of the control and experimental spot intensities were combined with the residual noise from each spot to obtain the filtered and adjusted expression values (Cui. et. al). Duplicate expression values from each array were combined, averaged and values from MAANOVA were subsequently subjected to significance analysis of microarray (SAM) data with the SAM package (Tusher. et.al). Significantly up- and down-regulated genes were identified based on a 1.5 cutoff threshold, a global false discovery rate lower than 5% (p<0.05). Six arrays representing a total of 12 replicates were analyzed between a treatment and a control.
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| |
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| Submission date |
Nov 16, 2011 |
| Last update date |
Nov 17, 2011 |
| Contact name |
zhe hou |
| E-mail(s) |
zhehou1018@gmail.com
|
| Phone |
612-578-2148
|
| Organization name |
University of Minnesota
|
| Department |
Food Science and nutrition
|
| Lab |
Diez&Sadowsky lab
|
| Street address |
1334 Eckles Ave
|
| City |
st. paul |
| State/province |
MN |
| ZIP/Postal code |
55108 |
| Country |
USA |
| |
|
| Platform ID |
GPL10291 |
| Series (1) |
| GSE33735 |
Escherichia coli Gene Expression Profiling in Response to the Interaction with Lettuce Rhizosphere |
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