Current Protocol for Spot-finding and Quantitation


  • E. coli arrays: (Sigma-GenoSys) Panorama E. coli gene arrays
  • Mouse arrays: (Clontech) Atlas Mouse 1.2 arrays

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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.
Copyright © 2009 The Board of Regents of the University of Oklahoma | Disclaimer
OU Bioinformatics Core Facility @ Advanced Center for Genome Technology | Credits | updated:19 Oct 2005