Tutorial for MasterPlex QT® v2.5 http://www.MiraiBio.com © 2007 Hitachi Software Engineering America, Ltd. All Rights Reserved.
Table of Contents Running MasterPlex QT using an existing template .............................................3 Marking and Grouping Wells...............................................................................13 Designating the Standard/Known Concentrations:..............................................21 Select the Standard Curve Model .......................................................................26 Utilizing the Virtual Plate Feature......................................................
Running MasterPlex QT using an existing template The following exercise will show how to run MasterPlex QT v2.5 using sample data that are included when the program is installed. When you start MasterPlex QT v2.5, you will see the plate wizard dialog box: Click the Next button: http://www.miraibio.
Select the Import a new plate option, and click Next: Click on the button with the folder icon to navigate to the .csv file. Navigate to C:\Program Files\HitachiSoft\MasterPlex QT 2.5\examples, and select the file titled IL5.csv. http://www.miraibio.
Next, click the Open button shown above. Click the Finish button. Click on the Template Manager icon shown circled above to select the template for this plate. http://www.miraibio.
Click on the M5PlexReplicates template, and click on the Load button. Please note how the wells are color-coded. • Copper = the background wells. • Blue = the standard wells. • Green = the unknown wells. • Yellow = the (optional) control wells. http://www.miraibio.
Also note the black borders that show how the wells are grouped. Please note that MasterPlex QT treats standard wells in a single group differently than background wells, unknown wells or control wells grouped together in a single group: • When standard wells are placed in one group, MasterPlex QT will use data from that group to create a standard curve for each analyte.
For this example, we will select the Five Parameter Logistics curve model, without weighting: Click on Assign Model. The following dialog box will show up: Click OK. http://www.miraibio.
Click Exit to leave the Model Equations dialog box. The following dialog box will then appear. Select Absolute Quantification, and click OK. http://www.miraibio.
You will then see the following dialog box: Now, from the drop-down pick-list, select Concentration/Fold Change. You can now toggle through the list of analytes on the left and view the concentration information for each analyte and each well. http://www.miraibio.
Note for the IL-10 analyte, some of the wells show a value <26.47. If we go to the dropdown pick-list, and select Standard/Independent Values, we see that the lowest known standard concentration for this analyte is 26.47. So, what does <26.
lowest point on the standard curve, we can infer that the concentration for that analyte is less than the concentration of the lowest point on the standard curve for that analyte. So, in the example above, we cannot use the model to calculate an estimated concentration, but we can infer that the concentration is less than 26.47 pg/mL.
Marking and Grouping Wells We’ll open the IL5.csv plate again, but this time, instead of using a template, we’ll mark the wells and enter the standard concentrations manually. Go ahead and close the plate we were working on in the above tutorial by clicking on the X in the top right-hand corner of the plate window: Now, click on the New icon to read in a .csv file produced by the Luminex machine (Note: If you are using build 163 or above, the New icon will not be present.
Navigate to C:\Program Files\HitachiSoft\MasterPlex QT 2.5\examples, and select the file titled IL5.csv. You will see the window shown below: This is the same plate we worked with before. If we did not have a template for this plate, we would need to mark the wells (as shown below). To mark wells, you highlight them, then right-click, and select how you want to mark them. http://www.miraibio.
We’ll mark A1 as background: Highlight the well, then right-click, and from the menu, select Mark Wells → Absolute Quantification → Background. The well will change color to copper (you may have to click on another well to change the highlighting to see the copper color indicating a background well). http://www.miraibio.
Now we will highlight B1 to H1, and mark them as Standard wells: Now right-click on the wells, and select Mark Wells → Absolute Quantification → Standards. http://www.miraibio.
The Standard wells will be grouped together and colored light blue: Now we’ll highlight A2, A3, A4, B2, B3 and B4, and mark them as a group of Unknown wells: http://www.miraibio.
Now right-click on the wells, and select Mark Wells → Absolute Quantification → Unknowns. The Unknown wells will be grouped together, and colored green: Note: Automatic grouping can be turned on or off by going to File → Preferences, and checking or unchecking Automatic Well Grouping. http://www.miraibio.
We can repeat this for the group C2, C3, C4, D2, D3, and D4. Highlight those six wells: Right-click on them, and designate them as Unknown: http://www.miraibio.
Repeat this for groups E2, E3, E4, F2, F3, F4 and G2, G3, G4, H2, H3, H4. http://www.miraibio.
Designating the Standard/Known Concentrations: We can now add the known concentrations for each analyte in the standard wells. The starting concentrations for each analyte are as follows: • • • • • IL-5 = 15,900 pg/mL IL-10 = 19,300 pg/mL IL-2 = 20,400 pg/mL IFN gamma = 5200 pg/mL IL-4 = 21,200 pg/mL The dilutions are 1:3. We can use the Auto fill feature in MasterPlex QT to quickly enter in the standard concentration values.
Next, right-click on the highlighted group, and select Auto fill from the menu. The Auto fill dialog box will appear: http://www.miraibio.
For analyte IL-5, the starting concentration is 15,900 pg/mL, and the dilution factor is 3 (since it is a 1:3 dilution). Note the various arrow keys – these are used to indicate the direction of the dilution; that is, the direction you would move as if you were doing the serial dilution in a 96-well plate, moving from most concentrated to least concentrated. The blue arrows and the red arrows allow you to specify the direction if the dilution spans more than one column or one row, respectively.
Now go back to the plate view, and select Standard/Independent Values from the pulldown pick-list. Now you can see the standard concentrations for IL-5. http://www.miraibio.
Note if you click on a different analyte, those standard concentration values are still zero. You will need to repeat this procedure for each analyte. Note that some kit manufacturers use the same starting concentration for all of the analytes in an assay. In this case, you can select in the Auto Fill dialog box the Fill in for all bead sets option, and it will use that starting concentration for all the analytes.
Select the Standard Curve Model Now we need to select the standard curve model to be used with each analyte. As we did before, we’ll use the 5-parameter logistic curve model. Right-click on one of the wells in your standard group, and select Assign Model Equation from the menu. http://www.miraibio.
Select Five Parameter Logistics for the model, and check Select this model for all analytes. Now click on Assign Model, and click OK in the confirmation dialog box: http://www.miraibio.
Now click Exit in the Model Equations dialog box. (If you see the following message: it just means that the choices you made here correspond to the default model assignment. Click OK.) The Select Calculation Type dialog box will appear. Select Absolute Quantification and click OK. At this point, you can click OK to perform the concentration calculations, as was done earlier in this tutorial. http://www.miraibio.
If you wanted to save this information in a template to apply to identical assays in the future, you will need to open the Template Manager: From here, you can click on Save, give the template a name, and save it for future use. http://www.miraibio.
Utilizing the Virtual Plate Feature The Virtual Plate feature allows you to combine data from multiple plate runs, perform cross-plate comparisons or expand the data analysis paradigm beyond the current 100 analyte per sample limit of the xMAP technology. The Virtual Plate feature also allows a researcher to combine the disparate data sets for a single patient sample. For this tutorial, we will stitch together 2 plates into one virtual plate. To follow along with this tutorial, you will need 3 sample files.
To open a Virtual Plate, please click on the Plate wizard icon. The Plate Wizard dialog will pop up. Click Next. Select the A virtual plate radio button and click Next. http://www.miraibio.
This dialog screen allows a user to define the size of the Virtual Plate they want to use. For this example, we will use the default size of 8 Rows and 12 Columns. Please click Finish. A new empty Virtual Plate will appear. http://www.miraibio.
Now, we are ready to merge our 2 sample files together. To begin, please click on the Virtual Pipette Tool. This handy tool will allow you to select, aspirate, and dispense the wells you would like to transfer. Highlight all the wells from the first sample file, right-click, and select Aspirate. http://www.miraibio.
To dispense the wells, highlight well A1 in the Virtual Plate, right-click and select Dispense. Note that the well you choose to dispense into will represent the upper-left most well from the selection of wells that you aspirated from. http://www.miraibio.
The Virtual Plate should now contain the data that is in the original sample file. http://www.miraibio.
Now, let’s do the same thing with the second sample file. Highlight all the wells from the first sample file, right-click, and select Aspirate. http://www.miraibio.
Highlight well E1 in the Virtual Plate, right-click and select Dispense. Note: If the number of bead regions and the bead region names are identical, MasterPlex QT will automatically map the names together in the Virtual Plate. The next example will show how to merge 2 plates with different bead region names and/or a different number of bead regions. Close all the existing plate files and load Sample1 and Sample3. Open a new Virtual Plate via the Plate Wizard.
Now, select and aspirate all the wells from Sample3. Right-click on well A1 in the Virtual Plate where you have your well from the first sample file and select Dispense. The Virtual Analyte Filter window will pop up. The New Analytes column lists the bead regions that you are trying to pipette into the Virtual Plate. The Assign Aliases column lists the bead regions that are already present in the well that you would like to map to.
The Virtual Plate now has all 10 bead regions merged together from the original 2 plate files. The Virtual Plate feature is a powerful and flexible tool that allows users to perform cross-plate analyses. http://www.miraibio.
Analyzing a single QuantigenePlex plate Note: This can also apply to similar one-plate relative quantification assays. 1. Open Plate: After launching MasterPlex QT, open the data file by clicking the Open icon or using File Open Plate. 2. Selecting bead regions that will be used for normalization: Right-click on the bead region that will be used to normalize the data with. Select Housekeeping. Repeat this step for all the bead regions that will be used for normalizing the data.
3. Designate well types: Select a well or group of wells and right-click. Navigate to the Relative Quantification submenu and select the appropriate well type: Assay Background, Control or Treatment. Repeat for all well types. http://www.miraibio.
4. Calculate results: Click the Calculate icon and choose 1 of the 3 available Relative Quantification analysis functions: http://www.miraibio.
a. Fold Change – This only will calculate the ratio between the Control and Treatment groups WITHOUT any data normalization. http://www.miraibio.
b. Fold Change – If the With normalization check box is ticked, it will perform a normalization step before the fold change calculation between the treatment and control groups. You have 3 options when normalizing: 1. Normalize by a single house keeping gene. 2. Normalize by the geometric mean of all the housekeeping genes selected in Step 1 if 2 or more genes were selected. 3. Normalized by a Constant value. c.
Other Features to Explore Another thing to explore is the Standard Curve Chart feature, which allows you to view your standard curves for each analyte. Click on the Open Standard Curves Chart button shown below. You can view the standard curve for each analyte. http://www.miraibio.
You can right-click on the chart, and select Set X-axis to log scale if you prefer this view. Click on the Standard Data tab. http://www.miraibio.
You can see Calculated, Expected, Residual, % Recovery and Background values for each analyte in each standard well. Also note that if you have outlier points here, you can designate them, so that they are ignored when you generate your standard curve. Another feature to explore is the Report Generator. After you have decided on which curve models you wish to use and performed the necessary calculations, you can click on the Report Generator icon shown below.
We’ll look at the Report per Unknown Replicates. Recall how grouping some unknown wells into a single group meant that you were designating those wells as replicate unknowns. Using the Report per Unknown Replicates, you can now view the mean, standard deviation and %CV for each group. Select the Report per Unknown Replicates option above, and click on the Generate button: http://www.miraibio.
You will see the following report: http://www.miraibio.
Note that you can then go to the Report menu and click on Save Report. You will then be presented with a number of options for saving the report (including PDF, HTML, text, and CSV [for Excel]). This Tutorial Guide just highlights some of the features of MasterPlex QT v2.5. Additional features can be found in the on-line manual by going to the Help menu in MasterPlex QT and selecting the Help option. On-line demonstrations can be found at: http://www.miraibio.com/online-demo/online-demos.
You can also contact MiraiBio with any questions you have at (650) 615-7600 (USA phone number) or by sending an email to support@miraibio.com. MiraiBio A Group of Hitachi Software 601 Gateway Blvd. Suite 100 South San Francisco, CA 94080 Telephone 1.800.624.6176 1.650.615.7600 Facsimile 1.650.615.7639 Trademark Acknowledgments MasterPlex is a trademark of Hitachi Software Engineering Co., Ltd. Luminex® is a registered trademark of the Luminex Corporation.
