|
| Status |
Public on May 15, 2010 |
| Title |
RetliCFN42_WT_vs_NifA_11ddi_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Nodules of eleven days post-inoculation, wild type
|
| Organism |
Rhizobium etli CFN 42 |
| Characteristics |
growth conditions: Symbiosis with bean 11 days post-inoculation
strain: CFN42
genotype/variation: wild type
|
| Biomaterial provider |
Nodules of Phaseolus vulgaris-Rhizobium etli
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Approximately 3 g of nodules were immersed in liquid nitrogen and macerated. Total RNA was isolated by acid hot-phenol extraction as described previously by de Vries et al.
|
| Label |
Cy3
|
| Label protocol |
10 ug of RNA was labeled with Cy3-dUTP using a CyScribe First-Strand cDNA labeling kit (Amersham Biosciences).
|
| |
|
| Channel 2 |
| Source name |
Nodules of eleven days post-inoculation, nifA mutant
|
| Organism |
Rhizobium etli CFN 42 |
| Characteristics |
growth conditions: Symbiosis with bean 11 days post-inoculation
strain: CFNX247
genotype/variation: nifA mutant
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Approximately 3 g of nodules were immersed in liquid nitrogen and macerated. Total RNA was isolated by acid hot-phenol extraction as described previously by de Vries et al.
|
| Label |
Cy5
|
| Label protocol |
10 ug of RNA was labeled with Cy5-dUTP using a CyScribe First-Strand cDNA labeling kit (Amersham Biosciences).
|
| |
|
| |
| Hybridization protocol |
Both Cy3 and Cy5 labeled cDNAs to be compared were mixed, dried to completion, and then resuspended in 45 ul of hybridization buffer UniHyb (TeleChem International INC). The mixture was hybridized at 42 C for 14 to 16 h. Hybridized arrays were washed with 2X SSC, 0.1% SDS at 42 C for 2 min, and, subsequently, with 0.1X SSC at room temperature for 2 min, and twice with 0.1X SSC at room temperature for 2 min.
|
| Scan protocol |
The array was scanned using a pixel size of 10 um with a Scan Array Lite microarray scanner (Perkin-Elmer, Boston, MA).
|
| Description |
Transcriptional profiling in symbiosis 11 days post-inoculation was compared between wild type strain and nifA mutant.
|
| Data processing |
Spot detection, mean signals, mean local background intensities, image segmentation, and signal quantification were determined for the microarray images using the Array-Pro Analyzer 4.0 software (Media Cybernetics, L.P). Microarray data analysis was performed with genArise software, developed in the Computing Unit of the Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico (http://www.ifc.unam.mx/genarise/). The Lowess normalization was applied using the genArise software. This software identifies differentially expressed genes by calculating an intensity-dependent z-score. It uses a sliding window algorithm to calculate the mean and standard deviation within a window surrounding each data point and defines a z-score where z measures the number of standard deviations that a data point is from the mean: zi = [Ri•mean(R)]/sd(R), where zi is the z-score for each element; mean (R) is the mean log ratio; Ri is the log ratio for each element; and sd(R) is the standard deviation of the log ratio.
|
| |
|
| Submission date |
Mar 04, 2010 |
| Last update date |
Mar 05, 2010 |
| Contact name |
Sergio Manuel Encarnación |
| E-mail(s) |
[email protected], [email protected]
|
| Phone |
777 3291899
|
| Fax |
777 3175094
|
| Organization name |
Universidad Nacional Autonoma de México
|
| Department |
Centro de Ciencias Genomicas
|
| Lab |
Genomica Funcional de Procariotes
|
| Street address |
Av. Universidad S/N
|
| City |
Cuernavaca |
| State/province |
Morelos |
| ZIP/Postal code |
62210 |
| Country |
Mexico |
| |
|
| Platform ID |
GPL10081 |
| Series (1) |
| GSE20638 |
Characterization of the NifA-RpoN Regulon in Rhizobium etli CFN42 in symbiosis using whole genome transcript analysis |
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