plant surface microbiology.pdf

29 Functional Genomic Approaches for Studies of Mycorrhizal Symbiosis 587
preparations or exposure time that might alter the signal intensity, normalize
the data obtained from different hybridizations by dividing the intensity
for each spot by the average of the intensities of all the spots present on
the filter, to obtain a centered, normalized value (Eisen et al. 1998).
8. It is important to take average values from multiple experiments to reduce
the variation from experiment to experiment. The final data can be analyzed
using Cluster and Treeview software (http://rana.lbl.gov) to obtain
more normalized data.
Note: You can also use k-means analysis and hierarchical clustering on the net
via the website: http://ep.ebi.ac.uk/EP/EPCLUST/ dedicated to statistical analysis
of gene expression data from macroarrays.
10.1 Data Analysis Autoradiography Images on X-ray Films
Note: If X-ray film is used to capture the image, it is important to do multiple
time exposures to obtain more reliable spot intensities for further analysis. It is
also important not to overexpose the X-ray film where the signal intensities are
not saturated. This will prevent the calculation of any subtle differences in the
expression levels.
1. Scan X-ray films at high resolution (1200x1200 dpi) using a scanner with
transparency adapter and save the images as TIFF files.
Note: These images take up a substantial amount of hard disk space (on average
25–30 MB).
2. Open the image in a quantification program such as One D-scan (Scanalytics
Inc., Fairfax,VA, USA).
3. Scale down the image to fit the screen using the scale and rotate option.
4. Draw a grid over the image using a preset size of 16 rows x 24 columns and
a numbering scheme to match the EST database.
5. Place the grid such that all spots are in the center of each cell. It is possible
to remove segments if artifacts or defects or over-intensity occur on
the image where the neighboring spots may overlap.
6. Calculate the spot intensity values using the volumes option in the analyzing
tool bar.
7. Calculate background using the boundary of each segment option, this
takes a value from each pixel bordering the cell and averages them to yield
the background value.
8. Using the volumes tool create a spread sheet of the data containing the
segment number with it’s background value and volume, along with high
and low values found within each cell.
9. Calculate the volume, or intensity, by adding the intensity of each pixel
within a given segment.
10. Transfer these data to Microsoft Excel (Microsoft Corp., CA) for further
manipulation.