GEArray Data Analysis Tutorial for Bio Research | SuperArray Bioscience Corp

1. GEArray Analysis *The step-by-step directions provided in this presentation are intended as a tutorial to guide the reader through the basic data analysis steps for the SuperArray Bioscience Corp GEArray gene arrays. The data handling choices in this tutorial (i.e. for background subtraction, normalization) should be taken as examples only, not as definitive rules for data handling.

2. Installing The Software • Go to www. SuperArray.com • Click on SUPPORT • Choose SOFTWARE • Log-in using Member e-mail and password • Choose DOWNLOAD SOFTWARE • Download GEArray Analyzer Software Program • Use provided link to download ScanAlyze for raw data extraction

3. Converting Array Image • Prior to loading your image into ScanAlyze, make the following conversions to your image using Photoshop: INVERTED GRAYSCALE 16 BIT/CHANNEL • Save your converted image as a.tiff file

4. Load Image Into ScanAlyze • Load .tiff file image into BOTH Channel 1 and Channel 2 • Click REDRAW to get image

5. Draw Grid • Click NEW GRID • Place # of columns and rows of your array • Change Spot Width and Spot Height. *Should not be bigger than 40 • Press Create

6. Adjust Grid • Use your mouse to move and adjust the grid to fit over the spots

7. Rotate Grid • Select By GRID • Highlight the grid* • Use the UP and DOWNred arrows to rotate the grid If your array image is not straight, you can rotate your grid to fit over the spots better. * To highlight grid: hold Shift key, then use mouse to click on grid. The change in grid color indicates that it has been highlighted.

8. Move Individual Grid Circle • Select by SPOT • Highlight Grid Circle* • Use Red Arrows to move your grid circle • To move another circle, UN-highlight the previous circle first and then repeat the steps. You may move individual grid circles to better fit over your array spots. *To highlight/UN-highlight circle: hold Shift key, then use mouse to click on grid circle. The change in circle color indicates that it has been highlighted/UN-highlighted.

9. Delete Grid • If you want to create a new grid, you will need to first delete the existing grid: • Select by GRID • Highlight grid* • Press DELETE GRID * To highlight grid: hold Shift key, then use mouse to click on grid. The change in grid color indicates that it has been highlighted.

10. Save Data • Save data… • …as a .dat file • Load next image file into ScanAlyze, make proper adjustments, and save. • After loading and saving all images, close ScanAlyze program

11. Open Files • Using Microsoft Excel, open the previously saved .dat files • Keep pressing NEXT until you • Finish.

12. Delete Rows • Delete rows 2 to 8, leaving only one text row (row 1).

13. Combine Files • Combine all files into one file, but place each set of data into a separate sheets Image 1 file Image 1 and 2 file Image 2 file

14. Save File *Make sure that the leftmost worksheet contains the control data control experimental *Remove any empty worksheets *A maximum of 10 worksheets can be analyzed at a time • Save file as a Microsoft Excel 5.0/95 Workbook (*.xls) file • Close Excel Program

15. Open GEArray Analyzer • Open GEArray Analyzer Program • Click New Analysis

16. Import File • Import File • Indicate Species • Indicate type of Array *(If you cannot find your array in the product menu, please go to the last slide “Import New Gene List”) • Choose Manual

17. Select Columns • Highlight CH1I column (column J) and press Select • Press Yes • Press OK

18. …Next • Press NEXT

19. Background Subtraction • Choose Minimum Value which will subtract the lowest measured value on the membrane from the values of all genes. *This will avoid negative values generated when using other value for subtraction.

20. Normalization Option • Normalize the gene expression values by a positive control gene (housekeeping gene) value • Pick the housekeeping gene that has an intermediate value (not too dark nor too light) • Press NEXT

21. Data Analysis Window • The Data Analysis window is a collection of multiple sheets representing the multiple steps during the Data processing: Raw Data, Consolidation, Subtraction, Normalization, Report, and Chart.

22. Raw Data • The Raw Data Sheet contains data imported from the data files and gene information obtained from the specified gene list file.

23. Consolidation Data • The Consolidation Data Sheet contains the averaged expression value of the positive and negative controls (which are spotted more than once). E.g. Average of the expression values of GAPD gene.

24. Subtraction Data • The Subtraction Data sheet contains gene expression values after minimum value subtraction. *APAF1 gene must have had the lowest expression value in the Exp2 membrane, and was therefore chosen by the program as the “minimum value”. That value was subtracted from the all the Exp2 values.

25. Normalization Data • The Normalization Data Sheet contains gene expression values afterminimum value subtraction and normalization. * The expression values were normalized to (divided by) the expression value of B-Actin.

26. Report Sheet • The Report Sheet includes processed data sets (minimum value subtracted & normalized) and ratio between the experimental and control data sets. *The Exp2/Exp1 column is the most important column because it shows the fold-change between the arrays.

27. Chart • The Chart Sheet contains visual representation of the analytic results.

28. Other Functions • Export Data button: To export all sheets to a Microsoft Excel file. • Filter Function: Apply a criteria to data comparison value set. • Exit button: To quit & exit program. • Print button: Print out current sheet in data analysis window. • Previous button: To go back to previous window.

29. Import New Gene List • Go to www.SuperArray.com • Click on SUPPORT • Choose SOFTWARE • Log-in using Member e-mail and password • Choose DOWNLOAD SOFTWARE • Download the New Gene List If you cannot find your array in the product menu, then you must import a new gene list. • Import the Gene List of your Array