Protocols: Creating New Quantification Protocols in ArrayVision

In most cases, this should not be necessary, since our standard ArrayVision protocol files should already be available within the program.

Procedure

  1. Run ArrayVision. (The most likely error at this point is that all network licenses are currently being used by someone else. If this happens, just wait and try again later.)
  2. Press the "Cancel" button on the initial wizard-choosing screen.
  3. Go to the menus and choose "Arrays -> Protocol manager...".
  4. From the Protocol Manager window menu, choose "File -> New".
  5. In the New Protocol window, make sure the "By wizard" choice is selected, then press the "OK" button.
  6. Press the "Next" button in the Welcome to the Protocol Wizard window.
  7. In the Method of Analysis window, select "Fluorescent" and "Expression", then press the "Next" button.
  8. In the Arrays and Channels window, set the number of arrays to 2 and the number of extra channels to 2. Make sure that "single template mode" is selected, then press the "Next" button.
  9. In the Channel Information window, select "Use file name as channel name" and press the "Next" button.
  10. In the Measure Selection window, choose "Volume" as the principal measure and "Median" for background. Then check the following selections: SD, Area, Position XY, Bkgd, S/N, and Flag. (You may need to scroll down to see them all. Volume and sVol will be selected automatically, and cannot be changed.) Then press the "Next" button.
  11. In the Template Organization window, check "Level 2", then press the "Next" button.
  12. In the Level 2 window, press the "Select image..." button to load a sample image. Use the standard file selection window to locate the image file. You will probably need to maneuver to another directory, and you will need to change the "Files of type" selection to "TIFF", since that is the format of the images produced by our scanner. (Do not select the similar looking "TIFF 5", which refers to an older version of the format.) Adjust the contrast or LUT map as described in the quantification protocol to make the spots easily identifiable.
  13. Our standard layout has 12 rows and 4 columns of grids. Enter those numbers in the Level 2 window. Use the layout ruler to measure the distance from the upper left corner of one grid to the upper left corner of the grid immediately below it. Enter this number as the interval between rows; it should be close to 4.5 mm.
  14. Repeat the procedure to measure the distance from the upper left corner of one grid to the upper left corner of the grid to its right. Enter this number as the interval between columns; this number is usually close to 4.4 mm. Thenpress the "Next" button.
  15. In the Level 1 window, we enter 10 rows and 10 columns to describe our standard grids. Use the layout ruler to determine the distance between adjacent spots vertically (interval between rows) and horizontally (interval between columns). Both of these numbers are usually close to 0.4 mm. Then press the "Next" button.
  16. In the Spot Parameters window, choose "Circle", and use the layout tool to determine the diameter of a typical spot. Note: ti change the size of the circle, press the Ctrl key and move the mouse without pressing any mouse buttons. You chould then click on several spots to confirm that the resulting size is correct; typically, it is about 0.33 mm. Press the "Next" button.
  17. In the Template Display window, select "Display Spots", "Spot outline", and "Display Level 2 boundaries", then press "Next".
  18. In the Labels window, select "Define structured labels". "Level 2" should be automatically selected, so you can just press the "Next" button.
  19. In the Level 2 labels window, select "Row/column numbers" from the drop-down list, then press the "Next" button.
  20. In the Level 1 labels window, select "Row/column numbers" from the drop-down list, then press the "Next" button.
  21. In the Label Display window, select "Level 2" and "Row/column headers", then press the "Next" button.
  22. In the Blanks window, leave everything blank and press "Next".
  23. In the Background window, select "Enable background subtraction" and "Corners between spots". Press the "Define" button. Set the size to 7 pixels, thne press the "OK" button and the "Next" button.
  24. In the Reference window, leave everything blank and press "Next".
  25. In the Anchor window, leave everything blank and press "Next".
  26. In the Post-Analysis Data Handling window, select "Save data file" and "Export data (text file: tab delimited)". Press the "Next" button.
  27. In the Protocol Wizard window, give your new protocol a meaningful name, and then press the "Finish" button.
  28. In spite of the previous button, you are still not finished. The Protocol Manager window will now open with your newly created protocol selected. Choose the menu item "Edit -> Modify". Decrease the sensitivity to 5 and the search radius to 5. Select the boxes for "Estimate element positions" and "Use extended search". Press the "Save" button. Now you are finished, and can proceed to the quantification protocol.

©2003-2010 The University of Texas MD Anderson Cancer Center
1515 Holcombe Blvd, Houston, TX 77030
1-800-392-1611 (USA) / 1-713-792-6161      Legal Statements