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VigeneTech

User’s Manual

Reverse Phase Protein Microarray

RPPM Module

VigeneTech Inc.

P.O. Box 272

Chelmsford, MA 01824

2006 Version 2.51

Page 41 of 42 1

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VigeneTech

IIntroduction to Reverse Phase Protein MicroArray Module

1Reverse Phase Protein Microarray

2Image and Dilution Data Analysis

IIImage Analysis with Control

1Image Layout

2RPPM Image Analysis

For a Regular and large Size RPPM image

Small Size ROI and Non Uniform Array

3Platemap File Defines Dilution

IIIDiluTion Data Analysis Process

1Configuration of Dilution Data Analysis

Open Dilution Option Dialog Box:

2Dilution Data Sub Window:

3View Dilution Data:

4Save and Open Dilution Data, .dxt file

IVFunction setup in Diltuion Data Analysis

1Background Subtraction and Negative Control

Regional Background Subtraction and Dust Removal

Background Subtraction from Negative Slide

Negative Control in Dilution Curve

2Normalization

Internal Reference Normalization:

Total Protein Normalization

Both Internal Reference and Total Protein Normalization:

Phosphate Protein Normalization

3Dilution Curve Fitting

Fitting Model:

Linear Range

5Automatic Fitting Model Selection

6y0 Intensity of Dilution Curve

7Reference Standard Unit (RSU) Curve

8Confidence Interval of

9Automatic and Manual Flag Outliers

10Replicates

11Mark Out Bad Curves

VReverse Phase Data Analysis Steps

Appendix A Dilution Curve Option Definition

Appendix B DIlution Data Output format

Appendix C Phoasphate protein normalication Data Output format

IIntroduction to Reverse Phase Protein MicroArray Module

1Reverse Phase Protein Microarray

.

Images of dilution curves in protein microarray contains various types of layouts and applications. For the same ID sample, there are a group of spots with different concentrations.

2Image and Dilution Data Analysis

The Reverse Phase Protein Microarray module is designed to provide a fully automated solution for post image or dilution data analysis. Combining with MicroVigene, the intergraded system provides users an end-to-end solution.

Dilution curve data Analysis

IIImage Analysis with Control

1Image Layout

The RPPM image is printed in such format that each sample or protein spotsare is distributed on the slide with different concentration; a collective of spots forms a dilution curve or replicates curves. By correctly, defining the linear range of those dilution curves,; it can accurately define the expression intensity and concentration of the sample or protein. The date output is the measurement of the group of spots in dilution curves instead the direct measurement for each of the spots alone.

CurrentCurrent most RPPM Image layoutsareis:

Low-density custom printed arrays

Non-uniform array in term of grid lay out, ROI, grid, and spot size and spacing

Multiple slides in the same image

Specific background, negative slide

Total protein or calibration slide

2RPPM Image Analysis

User’s Manual enu of MicroVigeneTM version 2.5 contains detailed configuration and operation information about image analysis.

For a Rregular and largecertainSsized RPPM image

MicroVigeneTM can automatically find the Region of Interest (ROI), place on the grid, segment spots pixel from the background, remove the dust, and quantify the intensity;, all in one mouse click operation.

Small Ssize ROI and Nnon Uuniform Aarray

Manually set up ROI frames theat first time for the same set of slides, select the Relocate Mode to “No relocate” at the configuration or the Options: General dialog box. Then MicroVigeneTM can automatic place the grid in each ROI, segment spots pixel from the background, remove the dust, and quantify the intensity;, all in one mouse click operation.

Note:See the tutorial in the MenuManual or on-line help for some examples on how to set up RPPM array, ROI frames, and template files.

3Platemap Ffile Defines Dilution

The RPPM Module uses platemap files to define the slide types, spots groups, spot control type, and dilution. See the User’s Manualenu for MicroVigeneTM v2.5 for detailed information about the Platemap Editor.

The default dDilution sets on the platemap file are on a ½ dilution and basedbased on the number of spots in each row.

Open Platemap Editor:

Select Platemap from the menu drop-down list of Application Menu: Edit | Platemap.

IIIDiluTion Data Analysis Process

1Configuration of Dilution Data Analysis

The process of dilution curve data analysis can be configured from the Dilution Option dialog box. Each functional area is configurable. The combinations of different selections can build the right process for various projects.

Open Dilution Option Dialog Box:

Select from dropdown list, Application Menu::Analysis | Plug in | Option on Dilution Data, as below, to open Dilution Option dialog box.

See the definition about each setting in the Appendix. For all supported functions and applications, see the next section of the Dilution Data Analysis and Set Uup.

2Dilution Data Sub Window:

Open Vview Ddilution Ccurve Ddata Wwindow:

Select the button on the Toolbar to open the view dilution data window as below. The dilution data panel is on the left and the image panel on right. The dilution data list is at the top and the dilution curve graphic area is at the bottomlow side of the panel. The data field for each curve in the list, the curve and data point on the curve, and spots on the image all associates together.

Dilution Data Sub Window

Toolbar in Dilution Data Sub Window :

Toolbar provides the short-cuts and new features for dilution data analysis:

confidence interval append horizontally montage Graphic

Save data to file Save data with reference Setup options

3View Dilution Data:

View Dilution Data and Curve Fitting Quality:

View Dilution Data List:

Top dilution data lList in the dilution data sub window all has most Dilution curve output information as in .dxt file. Check or un-check each measurement check box at the Show column in the Dilution Option dialog box to change the values measurements that are to be viewed. See Appendix B Output ******.dxt Format.

Display Curve Graphic and Highlight Curves on the Image:

From the List: Click any column fields in the list to show the dilution curves in the graphic area. Highlight all spot of the correspondent curves on the image.

Or click to highlight the left data list panel (first, then use the and keys to scroll up and down the curve list.

Spots on the Image:

Click on the data dot on the dilution curve graphic to highlight the spots on the image.

View Multiple Dilution Curve:

  • To sShow consecutive curves:

Hold down the Shift key and click any other dilution data from the list to show the consecutive curves. The colors highlighted in the list associate with the colors of the curves.

  • To sShow non-consecutive curves:

Hold down the Ctrl key and click any other dilution data from the list to show the selected curves. The colors also associated between the list and curves.

View Consecutive Curves View Selective Curves

Note:The rReference standard unit curve can be selected with any other curves only the dilution data points will beare the same as other sample curves.

View Linear Range Line:

Select a display option from the drop down list of Dilution Option dialog box to display the linear range line. See Liner Range, Non-linear curve fitting in section of IV.3.

View Confidence Interval:

Select linear or non-linear display confidence interval option from the drop down list, , on Dilution Data sub window to display the error estimate for curve fitting.

4Save and Open Dilution Data, .dxt file

The data shown in the dilution data list in the dilution curve window can be saved to a text file, .dxt file. See the Appendix for the file format.

To sSave dxt files:

Click button on Toolbar in Dilution Data sub window

Or

Select the Save Dilution Data menu drop-down list: Application Menu::Analysis | Plugin | Save Dilution Data.

To oOpen .dxt files in Microsoft Excel:

It can be opened in any text format application. It also can be opened in Microsoft Excel by right mouse clicking on the file name in Microsoft Explorer. Then click “Open with” to select Excel from the application list. Dilution Data Analysis and set up

IVFunction setup in Diltuion Data Analysis

1Background Subtraction and Negative Control

The RPPM Module provides three levels of background subtraction. It is a spots based operation. Based on the application, the background process can be any combination of these three types:.

Regional Background Subtraction and Dust Removal

UseUsingthe MicroVigeneTM propitiatory regional background subtraction algorithm to remove the background from the spot’s signal. This process also can automatically remove the dust in the spots. See the User’s ManualenuofMicroVigeneTM v. 2.0.

Background Subtraction: meat_net, median_net, vol_net

No Background Subtraction: meat_Total or median_Total, Vol_Total

To set up Intensity type at the Dilution Options dialog box, by selecting itone of in the dropdown list:

Negative ControlBackground Subtraction from Negative Slide

Use the negative slide to do spots by spots intensity subtraction to remove the specific background or systematic background in the sample slide. Negative slides can be in a separate file or in the same image files:

Separate Negative Slide Image:

ANegative negative slide or negative slides are in a different image file than sample slides.

Create a Negative Slide Date File, .nxt:

Click the Save Negative Slide button in the Dilution Options dialog box, to save a negative slide file, .nxt from a negative slide image. It can be subtracted by other sample slides for negative slide background subtraction. This file will be save in the same folder as its image file is. In this file, each spots has an out put intensity value.

Subtract Negative Slide Data file, .nxt:

When a sample slide image file needs to have a negative slide image background subtraction, in theat Dilution Options dialog box, click the button to open the file browser. Select the pre-saved .nxt file to following text field.

Negative Slide in the Same Image:

ANegative negative slide or slides are in the same image file as sample slides.

Defininge Negative Slide in a Platemap File:

Select the Application Menu::Edit|Platemap to open the Platemap Editor. Then update the Group column with the negative slide identifier, neg. or use Microsoft Excel to update.

Set-up Internal Negative Slide Subtraction

:

At the Dilution Options dialog box, select the internal from the negative slide drop-down list:

NNegative Control in Dilution Curve

With or without the predefined negative control spots in the platemap file, the RPPM Module can do the negative control background subtraction.

Negative Control Spots:

Define in Platemap File:

Select the Application Menu:: Edit|Platemap to open the Platemap Editor. Click on the Control column (in the spot’s row field, not the title field). From the control type drop-down list, select negative to update with the control type, n.

Or use Microsoft Excel to , update the control column for negative control spots with a number of “4”.

Set-up Negative Control Subtraction:

Check the Negative Control check box in the Dilution Options dialog box:

Background Subtraction without Negative Control Spots:

When the Negative Control Check box is checked, but there is no negative control spots defined in the platemap file, it will detect the spot with the lowest intensity in the curve as the negative control spot. All the spots in the same curve will be subtracted by this lowest spot intensity.

2Normalization

The RPPM provides normalization options at a dilution curve level: normalizations in type of internal reference and total protein normalizations.

Note: MicroVigeneTM supports spots level normalizations.

Internal Reference Normalization:

Positive control:

Using Use the positive control curve or curves defined in the platemap file to do the normalization inside of each image, slide, or group of ROIs. The positive control curve will be normalized to 1000.

Define in Platemap File:

Select the Application Menu:: Edit|Platemap to open the Platemap Editor. Click on the Control column (in the spot’s row field, not the title field). From the control type drop-down list, select positive or positive-ref to update with the control type, n.

Or use Microsoft Excel to, update the control column for the positive control curve with a number of “1” or positve-ref for “3”.

Set-up Positive Control Normalization:

Select the Internal Reference in the Normalize dropdown list in the Dilution Options dialog box:

Global Normalization:

If the Internal Reference is selected in the above Normalize dropdown list and there are no positive control spots or curves defined in the platemap file, a global normalization willwill be performed ats a group base. Normalization will use the average y0 value, over one slide or over all ROIs in the same group, to do the normalization. The average y0 will be normalized to 1000.

Define Normalization Group in Platemap File:

Select the Application Menu:: Edit|Platemap to open the Platemap Editor. Enter the same ID name for all ROIs in the same normalization group:

Or use Microsoft Excel to, update the slide column for the same ID for an internal global normalization.

Three Level Group Based Normalization:

The RPPM module can perform any type of internal reference normalization between iImages, sSlides in the same image, ROIs in the save image, or groups of ROIs in the same slide or in the different slides images.

Total Protein Normalization

Use the total protein image dilution data file to do the total protein normalization.

Separate Ttotal Pprotein Ffile:

ANegative total protein slide or slides are in a different image file than sample slides.

Selected at the Total Protein… field. Normalize with separate Total protein reference file, _ref.dxt.

Create a Total Protein Data File, .*_ref.dxt:

Click the Save Total Protein button in the Dilution Options dialog box, to save a total protein data file, *_ref.dxt from a total protein image. It can be subtracted by other sample slides for negative slide background subtraction. This file will be saved in the same folder as its image file is. In this file, each curve has an out put value.

NormalizeTotal Protein Data file, *_ref.dxt:

When a sample slide image file needs to have total protein normalization, in theat Dilution Options dialog box, click the button to open the file browser. Select the pre-saved _ref.dxt file to following text field.

Select Total ProteinNormalization:

Select the Total Protein in the Normalize dropdown list in the Dilution Options dialog box:

Total Protein Slide in the Same Image:

ANegative Positive slide is in the same image file as the sample slides.

Defininge Total Protein Slide in a Platemap File:

Select the Application Menu::Edit|Platemap to open the Platemap Editor. Then update the Group column with the total protein slide identifier, ref or use Microsoft Excel to update.

Set-up Total Protein Normalization

:

1)At the Dilution Options dialog box, select the internal from the Totel Protein drop-down list:

2)Select Total Protein in the Normalize dropdown list in the Dilution Options dialog box:

Both Internal Reference and Total Protein Normalization:

Use theing internal reference normalization first then thedoes total protein Normalization.

Select Both in the Normalize dropdown list in the Dilution Options dialog box:

Phosphate Protein Normalization

Phosphate Protein Normalization is the normalization between Phosphate protein slide with its non-phosphate protein slide.

Step 1: Load and analyze non-phosphate protein slide or slides image file. Then save the dilution data in .dxt file. Close the sub window.

Step 2: Load and analyze the phosphate protein slide or slides image file.

Step 3: Normalize the non-phosphate protein slide:

  1. Click button on Toolbar in Dilution Data sub window to load non-phosphate protein .dxt file:
  1. Click open button to normalize the non-phosphate protein and save the file with the in such name convention: PhosphateName_NonPhosphatName.dxt.

Note: Phosphate and non-phosphate protein image file should have same number of data points.

3Dilution Curve Fitting

4RPPM Module version 2.0 support linear two parameter curve fitting and non-linear four parameter curve fitting in linear or log scale. Choose any one in this list to properly suite your dilution curve. Reach the best curve fitting result to the experemente data.