NSF Rice Oligonucleotide Array Validation
The 45K array was validated using RNA harvested from light/dark grown rice plants
For a detailed description of the experiment used to validate the 45K array design, download the Validation of NSF supported rice 45k array with light vs. dark experiment (pdf, 164K).
The 20K array was validated using RNA harvested from light/dark grown rice plants
Error Rate
1. The error rate was evaluated using 217 hygromycin spots. The 20K chip has 217 spots corresponding to the hygromycin resistance gene, which are randomly spotted. These spots can be used as positive controls for transgenic samples, and also as a negative control for nontransgenic samples.
Example 1. Positive control; NH1ox transgenic plants vs wild-type.
| Slide | 1 | 2 | 3 | 4 | 5 | 6 |
| Number of hph spots > 2 fold | 217 | 217 | 217 | 217 | 217 | 217 |
| Ratio (%) | 100 | 100 | 100 | 100 | 100 | 100 |
| Total hph spots | 217 | 217 | 217 | 217 | 217 | 217 |
Example 2. Negative control; light vs. dark-grown non-transgenic seedlings.
| Slide | 1 | 2 | 3 | 4 | 5 | 6 |
| Number of hph spots > 2 fold | 2 | 0 | 1 | 1 | 1 | 1 |
| Ratio (%) | 0.92 | 0.00 | 0.46 | 0.46 | 0.46 | 0.46 |
| Total hph spots | 217 | 217 | 217 | 217 | 217 | 217 |
Reproducibility
2. The reproducibility was tested using dye swapping.
| Dark (cy3 green) light (cy5 red) | Light (cy3 green) dark (cy5 red) |
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Correlation coefficient and scatter-plot between replicates:
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Sources of Variation
3. Sources of variation were evaluated (using ANOVA, Rocke et al.) In this example, the treatment factor was the major source of variation:
Gene Ontology Analysis
4. Gene Ontology (GO) analysis was performed.
Summary of GO annotation for the 20K oligo set:
| GO Terms | 0.1% FDR | 5% FDR | Total |
| Biological Process | 1,038 (37.5%) | 2,272 (34.8%) | 5,645 (27.9%) |
| Cellular Component | 558 (20.2%) | 1,233 (18.9%) | 3,015 (14.9%) |
| Molecular Function | 1,400 (50.6%) | 3,051 (46.8%) | 7,227 (35.7%) |
| All GO-terms | 1,530 (55.3%) | 3,330 (51.0%) | 7,967 (39.4%) |
| Light/dark Microarray | 2,767 (100% ) | 6,525 (100%) | 21,120 (100%) |
e.g. Photosynthesis. Result of light/dark microarray experiment for genes involved in photosynthesis. 14 of 19 genes were differentially expressed in terms of p-value (FDR) and fold change. TIGR ID was the name of gene model in the TIGR Rice Annotation release 3. FDR was the false discovery rate (p-value) developed by Rocke (2004).
| TIGR ID | Biological Process | Putative Function | FDR | Log2L/D |
| 11673.m03640 | Photosynthesis | Chlorophyll A-B binding protein | 1.05E-07 | 6.068103 |
| 11674.m03347 | Photosynthesis | Chlorophyll A-B binding protein | 1.05E-07 | 6.855869 |
| 11674.m00949 | Photosynthesis | Photosystem II PsbR | 1.09E-07 | 6.849156 |
| 11673.m03790 | Photosynthesis | Chlorophyll A-B binding protein | 1.32E-07 | 6.155692 |
| 11681.m01619 | Photosynthesis | Chlorophyll A-B binding protein | 1.32E-07 | 7.033996 |
| 11667.m04019 | Photosynthesis | Chlorophyll A-B binding protein | 1.62E-07 | 7.028789 |
| 11669.m05717 | Photosynthesis | Photosystem I F subunit | 1.91E-07 | 4.929572 |
| 11673.m00461 | Photosynthesis | Photosystem I psaG / psaK | 2.12E-07 | 6.161693 |
| 11668.m05158 | Photosynthesis | Chlorophyll A-B binding protein | 4.03E-07 | 3.17475 |
| 11667.m07138 | Photosynthesis | Photosystem II PsbW | 6.77E-07 | 2.950762 |
| 11667.m05117 | Photosynthesis | Chlorophyll A-B binding protein | 1.24E-06 | 2.084434 |
| 11669.m04812 | Photosynthesis | Ferredoxin | 3.79E-06 | 1.590981 |
| 11667.m00476 | Photosynthesis | Triosephosphate isomerase | 7.64E-06 | 1.418197 |
| 11673.m00448 | Photosynthesis | Photosystem PsbR | 5.87E-03 | 1.417425 |
| 11667.m06239 | Photosynthesis | Triosephosphate isomerase | 4.58E-02 | 0.17687 |
| 11669.m06319 | Photosynthesis | Ferredoxin | 7.04E-02 | -0.15064 |
| 11669.m04559 | Photosynthesis | Ferredoxin | 1.05E-01 | 0.191392 |
| 11682.m03483 | Photosynthesis | Ferredoxin [2Fe-2S] | 1.05E-01 | 0.109432 |
| 11670.m03233 | Photosynthesis | Ferredoxin [2Fe-2S] | 9.33E-01 | -0.01316 |
Differentially Expressed Genes
5. Differentially expressed genes were identified using LMGene (Rocke et al.) The list of differentially expressed genes in the light vs. dark experiment with both log2 ratios and p-value are given below:
References
Chern MS, Fitzgerald HA, Canlas PE, Navarre DA, and Ronald PC. 2005. Over-expression of a Rice NPR1 Homologue Leads to Constitutive Activation of Defense Response and Hypersensitivity to Light. MPMI. 18. (6): 511-520.
Rocke DM. 2004. Design and Analysis of Experiments with High Throughput Biological Assay Data. Sem. Cell Dev. Biol., 15, 708-713.
Last modified: Monday, 06-Nov-2006 16:28:12 EST