|
| Status |
Public on May 16, 2011 |
| Title |
Bj_paraquat fulminant shock vs. no treatment_Rep1_dyeswap |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Paraquat fulminant shock treatment_Replication 1
|
| Organism |
Bradyrhizobium diazoefficiens USDA 110 |
| Characteristics |
'strain: USDA 110', 'Treatment type: paraquat', 'Treatment concentration: 5 mM', 'Treatment time: 10 minutes', 'Medium: Arabinose-gluconate'
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Standard hot-phenol method
|
| Label |
cy3
|
| Label protocol |
30 ug of total RNA was used for cDNA synthesis with superscript III reverse transcriptase and random hexamers (Invitrogen Corp.). After digesting the remaining RNA by RNase H (Fermentas), cDNA was purified using a Microcon YM-30 column (Millipore) and concentration was determined using a Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). Five micrograms of cDNA were used for labeling with either cy3 or cy5 (Armersham) and unincorporated dyes were removed using a Qiaquick PCR purification Kit (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
No Treatment Replication 1
|
| Organism |
Bradyrhizobium diazoefficiens USDA 110 |
| Characteristics |
'strain: USDA 110', 'Treatment type: None', 'Medium: Arabinose-gluconate'
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Standard hot-phenol method
|
| Label |
cy5
|
| Label protocol |
30 ug of total RNA was used for cDNA synthesis with superscript III reverse transcriptase and random hexamers (Invitrogen Corp.). After digesting the remaining RNA by RNase H (Fermentas), cDNA was purified using a Microcon YM-30 column (Millipore) and concentration was determined using a Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, DE). Five micrograms of cDNA were used for labeling with either cy3 or cy5 (Armersham) and unincorporated dyes were removed using a Qiaquick PCR purification Kit (Qiagen).
|
| |
|
| |
| Hybridization protocol |
Both cy3 and cy5 labeled cDNAs to be compared were mixed, dried to completion, and then resuspended in 70 ul of preheated hybridization buffer (nuclease free water:formamide:20X SSC:1% SDS = 4:2.5:2.5:1) at 42 C. After adding 0.7 ul salmon sperm DNA (10 mg/ml) to prevent non-specific binding to the array, the mixture was hybridized at 42 C for 16 to 18 h. Hybridized arrays were washed with 1X SSC, 0.2% SDS at 42 C for 6 min, and, subsequently, with 0.1X SSC, 0.2% SDS at room temperature for 6 min, and twice with 0.1X SSC at room temperature for 3 min.
|
| Scan protocol |
The arrays were scanned using an Axon GenePix 4000B scanner (Molecular Devices Corp., Sunnyvale, CA). Signal intensities were analyzed using GenePix Pro 6.0 software (Molecular Devices Corp., Sunnyvale, CA).
|
| Description |
Transcriptional profiling of paraquat treated cells compared to non-treated cells.
|
| Data processing |
Signal intensities were normalized for spot and slide abnormalities using spatial lowess. Lowess adjusted data were subsequently analyzed by mixed effect microarray analysis of variance (MAANOVA) (Kerr et al. 2000). The resulting variety-by-gene interaction (VG) values were combined with the residual noise from each spot to obtain the filtered and adjusted expression values (Kerr et al. 2000; Kerr and Churchill 2001). Both lowess and MAANOVA are part of the R/maanova microarray statistical analysis package (http://www.jax.org/staff/churchill/labsite/). Expression values from two technical replicates in each array were combined and averaged. Significant genes were selected based on a 1.5-cut off threshold with a false discovery rate lower than 5%.
|
| |
|
| Submission date |
Dec 21, 2010 |
| Last update date |
May 16, 2011 |
| Contact name |
Woo-Suk Chang |
| E-mail(s) |
[email protected]
|
| Phone |
1-817-272-3280
|
| Organization name |
University of Texas at Arlington
|
| Department |
Biology
|
| Lab |
Woo-Suk Chang's Lab
|
| Street address |
501 S. Nedderman Dr. , Life Science Building 216
|
| City |
Arlington |
| State/province |
TX |
| ZIP/Postal code |
76019 |
| Country |
USA |
| |
|
| Platform ID |
GPL5341 |
| Series (2) |
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