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Current Protocol for Spot-finding
and Quantitation
Arrays:
- E. coli arrays: (Sigma-GenoSys)
Panorama E. coli gene arrays
- Mouse arrays: (Clontech)
Atlas Mouse 1.2 arrays
Download: (note: for non-IE browers, hold down shift key then select)
TIFF Image Files:
- Exposed phosphorscreens are scanned at 100 micron
pixel size on a Storm 820 (Molecular Dynamics). The Storm creates
.gel files that can be viewed in ImageQuant (http://www.amershambiosciences.com).
- Gel files are organized and put into folders that correspond with the experiment (Don's folder, Angela's folder, etc). Each personal folder contains folders called "Analysis, Crunched, and Original Images. Save the original .gel image in the original images folder.
Spot-finding and Image Quantitation:
- Array Vision, version5.1, is a software analysis
tool written by Imaging Research , Inc. (www.imagingresearch.com)
and made available from Amersham-PharmaciaBiotech (http://www.amershambiosciences.com)
- The analysis protocol, including the grid template
can be downloaded. The template must be downloaded and saved in
the Array Vision protocol editor before it can be implemented
in the analysis wizard. Open “protocol editor”, click File, Open
4.0 template, select the gride file (*grid.sg), and click Open.
Next, click File, Save as protocol…, enter protocol name, and
click Save. These operations will save the template and analysis
protocol for future use in the analysis wizard.
- Important features of the customized template, ecoligrid.sg,
include template organization in the layout dialog (3 levels:
3 x1, 16 x 24, 4 x 4), blanks defined in level 1, and labels defined
in all 3 levels (ie. "F1:A-1:1" to indicate field:column-row:spot);
background defined in the measures dialog as a 5 pixel "strip"
surrounding entire template; and alignment parameters defined
in the alignment dialog.
- Important features of the customized template, mousegrid.sg,
include template organization in the layout dialog (1 level of
32 x 46), blanks defined in level 1 between 6 fields of array,
and labels defined by array location (ie. "A01a" to indicate field:column:row);
background defined in the measures dialog as corners between spots;
and alignment parameters defined in the alignment dialog.
- When Array Vision is opened, a dialog box will open
with three choices: analysis wizard, automation wizard, and open
a data file. Choosing "Analysis Wizard" is the easiest way to
analyze .gel images. When the "Next" button is clicked, a variety
of protocols will be available to choose from; be sure that the
“ecoligrid” protocol is selected.
- To open the data file to be analyzed, "Add" the
desired image file, and click "Done" when finished.
- The image is now in ArrayVision, but might not be
visible due to the contrast of the image. After ArrayVision has
loaded the image, it will display an X/Y coordinate graph in the
center of the screen. This graph is used to adjust the contrast
in the image. In most cases clicking the “Auto Contrast” button
located at the bottom of the X/Y coordinate generates an image
that has an optimal contrast for analysis.
- Next, he template needs to be correctly aligned
over the image. There are many tools on this screen to help with
this process. First, the entire template is moved over the array
image by a click and drag process using the green “handle” on
the upper left corner of the template, making sure that the upper
left elements are aligned with the corresponding spots. The current
version of Array Vision has a tough time aligning the template
over all 3 fields, so it is best to align each field separately.
An individual field is selected by clicking to the left of the
top row, then holding the shift key while clicking on the bottom
row. The rough alignment of the field grid is accomplished manually,
in five steps:
- align upper left spots
- click and drag bottom border of grid to align with bottom left spots
- click and drag right border of grid to align with upper right spots
- click and drag upper right “handle” to rotate grid.
- repeat steps 2 and 3 such that each grid element is roughly positioned over each spot
- Click the “Auto Align” button. The alignment parameters
should already be optimized such that the grid will be perfectly
aligned on the field. This process is repeated independently for
the three fields and with practice should take less than 3 minutes.
- The wizard will ask to export the data generated from the image. Don't export at this step. Choose close, and then look at the table.
- For E. coli array, click the button in the right hand corner that puts the data in a "primary/secondary" table format. The data will be organized with Level 2 and 3 array coordinates in rows and Level 1 coordinates in columns. This is what needs to be exported.
- For mouse array, keep data in "Target table" format.
- Select File, Export. Export to the "Export" folder, but BE SURE to save as an .XLS file.
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