|
| Status |
Public on Jul 21, 2015 |
| Title |
Replication 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Bj110
|
| Organism |
Bradyrhizobium diazoefficiens USDA 110 |
| Characteristics |
genotype/variation: Bj110
growth condition: Free-living cells grown under desiccation stress (27% RH)
|
| Growth protocol |
The bacterial cells were grown until mid-log phase (OD = 0.8) and filtered on 0.45 micrometer filter papers. The filter papers containing the bacterial cells were exposed to desiccation condition (27% RH) for 24 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by the standard hot phenol extraction method.
|
| Label |
Cy3
|
| Label protocol |
10 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 (Fisher Scientific). Four micrograms of cDNA were used for labeling with either cy3 or cy5 (GE Healthcare) and unincorporated dyes were removed using a Qiaquick PCR purification Kit (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
BjΔaceA
|
| Organism |
Bradyrhizobium diazoefficiens USDA 110 |
| Characteristics |
genotype/variation: BjΔaceA
growth condition: Free-living cells grown under desiccation stress (27% RH)
|
| Growth protocol |
The bacterial cells were grown until mid-log phase (OD = 0.8) and filtered on 0.45 micrometer filter papers. The filter papers containing the bacterial cells were exposed to desiccation condition (27% RH) for 24 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by the standard hot phenol extraction method.
|
| Label |
Cy5
|
| Label protocol |
10 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 (Fisher Scientific). Four micrograms of cDNA were used for labeling with either cy3 or cy5 (GE Healthcare) 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 4200A scanner (Molecular Devices Corp., Sunnyvale, CA). Signal intensities were analyzed using GenePix Pro 6.0 software (Molecular Devices Corp., Sunnyvale, CA).
|
| Description |
Reference:Test=Ch1:Ch2
|
| 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. 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 |
Jun 18, 2015 |
| Last update date |
Jul 21, 2015 |
| 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 (1) |
| GSE69999 |
Characterization of AceA regulons in Bradyrhizobium japonicum under desiccation stress |
|
