|
| Status |
Public on Nov 23, 2010 |
| Title |
PCA_donorvscontrol_potentiostat_BR2A |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Geobacter sulfurreducens current-consuming biofilm; experimental condition
|
| Organism |
Geobacter sulfurreducens |
| Characteristics |
growth protocol: current-consuming biofilm
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TE-sucrose hot-phenol extraction method, subsequent DNAse treatment with Ambion DNA-free protocol.
|
| Label |
Cy5
|
| Label protocol |
Micromax ASAP direct RNA labeling protocol
|
| |
|
| Channel 2 |
| Source name |
Geobacter sulfurreducens no-current biofilm; control condition
|
| Organism |
Geobacter sulfurreducens |
| Characteristics |
growth protocol: no-current biofilm (control)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TE-sucrose hot-phenol extraction method, subsequent DNAse treatment with Ambion DNA-free protocol.
|
| Label |
Cy3
|
| Label protocol |
Micromax ASAP direct RNA labeling protocol
|
| |
|
| |
| Hybridization protocol |
Combimatrix 100 ul volume cap.
|
| Scan protocol |
Slide was scanned using GenePix software and a GenePix 4000b scanner
|
| Description |
total RNA was directly labeled with the Micromax ASAP labeling kit, using 10 ug total RNA for each sample.
|
| Data processing |
The raw total intensities were treated as follows to generate the M Log2 ratios for this work. Background signal was calculated from a set of negative control spots incorporated into the array. As the negative control sequences are not design specific (ie. are found in all of the ESOAs independent of the organism being sampled) some will inevitably demonstrate strong cross hybridization with the sample of interest. To avoid this issue the negative control spots were ranked according to their total intensity. The average of the lowest 30% was calculated. Background signal was calculated as this mean + 2 standard deviations. The background signal was then subtracted from the total intensity for each spot. Probes whose signal was not greater than zero after background subtraction were omitted from further analysis. For the remaining data, the logged ratios [M=log2 (Ex/Ct)] are then calculated and Lowess global normalization is performed. During the data preprocessing, M vs. A plots before and after normalization, and side-by-side box plots for all arrays are used to assess array quality.(3) LIMMA mixed model analysis (R-package LIMMA(2))is applied to the normalized logged ratios to identify differentially expressed genes. Specifically, biological replicates are treated as a randomized block to allow for modeling correlations within them. LIMMA calculates a moderated t statistic for each probe, which is the ratio of the estimated mean logged intensity ratio and the Empirical Bayes estimate of its standard error. The P-value of each oligo is then corrected for multiple comparisons according to Benjamini and Hockberg's procedure(1) to control the false discovery rate. Oligos are ranked according to the adjusted P-values and were called differentially expressed if the adjusted P-values are < 0.001. Note that multiple oligonucleotide probes from the same gene are analyzed separately and a gene is called differentially expressed if at least half of its probes are called differentially expressed.
1. Benjamini, Y., and Y. Hochberg. 1995. Controlling the False Discovery Rate - a Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B-Methodological 57:289-300.
2. Smyth, G. K. 2004. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3:Article3.
3. Smyth, G. K., and T. Speed. 2003. Normalization of cDNA microarray data. Methods 31:265-73.
|
| |
|
| Submission date |
Nov 23, 2009 |
| Last update date |
Nov 24, 2009 |
| Contact name |
Sarah Strycharz |
| E-mail(s) |
[email protected]
|
| Phone |
202-767-3822
|
| Fax |
202-334-2759
|
| Organization name |
Naval Research Laboratory
|
| Department |
CBMSE
|
| Lab |
Tender
|
| Street address |
4555 Overlook Ave.
|
| City |
Washington |
| State/province |
DC |
| ZIP/Postal code |
20375 |
| Country |
USA |
| |
|
| Platform ID |
GPL4897 |
| Series (2) |
| GSE19149 |
Expression Analysis of Geobacter sulfurreducens Current-Consuming Biofilm versus No-Current Biofilm |
| GSE19158 |
Geobacter sulfurreducens biofilm transcriptomics |
|
