Maize Microarray Data Management
study project A project "Maize" was created in GeneDirector to archive and manage the entire microarray data for the published study. The project contained eight subfolders, namely, PROBE, PLATE, ARRAY, SAMPLE, HYBRIDIZATION, SCANNED_ARRAY, QUANTIFIED_ ARRAY, and PROCESSED_DATA.
creation of array design An array design "Maize" was created in GeneDirector that contains the array layout for maize data. Clicking on this array design displays the probes printed on the array. This array design is displayed in the "Array Designs" folder of the "My Data" database.
arrays within project Based on the array design, arrays were created and stored in the GeneDirector database corresponding to the study experimental design. There are three sets of individual comparisons, namely, Epidermal vs. Vascular, Epidermal vs. Coleoptile, and Coleoptile vs. Vascular.
Four arrays were created for each pair comparison in which two arrays are two biological replicated flip-dye experiments. Clicking on any array, like the array design, displays the probes present on the array beneath its panel. All the arrays used in the project are displayed in the ARRAY folder in the GeneDirector application.
samples and study protocols Sample annotations were entered into GeneDirector via the sample tracker to store the study sample information. For each array, GeneDirector tracks three biological sample types, BIOMATERIAL, RNA SAMPLE, and LABELED SAMPLE.
All the biological samples used in the project are displayed in the SAMPLE folder in the GeneDirector application. The experimental protocols, including RNA extraction, amplification, and labeling, were associated with their corresponding samples and stored in the database.
array hybridizations Hybridization entities were created for the four different arrays in each of the three sets of experimental pair comparisons. Hybridizations were created using the labeled samples and arrays.
All the hybridizations used in the project are displayed in the HYBRIDIZATION folder in the GeneDirector application. These hybridizations were associated with their protocols and are displayed in the hybridization editor window.
scanned images Scanned arrays were created for every hybridized array by importing their corresponding Cy3 and Cy5 images into the GeneDirector database. All the scanned arrays used in the project are displayed in the SCANNED_ARRAY folder in the GeneDirector application.
image analysis These scanned arrays were quantified with GeneDirector's image analysis module ImaGene. Using proprietary segmentation algorithms, ImaGene converts the pixel values of every spot on the scanned array into its numeric quantification value along with the appropriate flagging for the unusual spots. The ImaGene module of GeneDirector also offers automated batch analysis of the scanned arrays.
The quantifications of the scanned arrays were stored in the GeneDirector database and displayed in the QUANTIFIED_ARRAY folder. Selecting a quantified array in the relationship viewer helps to view the relationship of this array with all other objects in the database, e.g., the array design and array used.
statistical analysis The quantified arrays stored in the GeneDirector database were launched into GeneSight for knowledge discovery. The data analysis module GeneSight, embedded within GeneDirector, is a bioinformatics software solution that offers exploratory data mining and confirmatory statistical analyses tools to obtain biological insights from the complex and high dimensional microarray experiments.
Data were paired.These data were transformed in the following sequence:
Local background correction for every spot individually
Omit flagged spots (empty, poor, and negative)
Floor low expression values to 20
Log base 2
Difference across array channels
Combine within array replicates
An individual student's t-test was computed for each of three sets of experimental pair comparisons, namely, Vascular and Coleoptile, Epidermal and Vascular, and Epidermal and Coleoptile. The genes found significant at p<0.001 were partitioned (filtered) out to generate reports.
The common genes among the three sets of experimental pair comparisons were determined using the "Intersection" tool of GeneSight.
These analyzed data were stored in the database and displayed in the PROCESSED_DATA folder in the GeneDirector application. These processed data can be re-launched into GeneSight for review and/or further analysis.