TUTORIAL MasterPlex ReaderFit A H I T A C H I S O F T W A R E C O M P A N Y For Research Use Only 601 Gateway Blvd. Suite 100 South San Francisco, CA 94080 TELEPHONE 1.888.615.9600 (toll free) 1.650.615.7600 FACSIMILE 1.650.615.7639
TRADEMARKS Microsoft is a registered trademark of Microsoft Corporation. COPYRIGHT 2009 Hitachi Software Engineering America, Ltd. All Rights Reserved. Ver P122009-29 MasterPlex ReaderFit www.miraibio.com i
TRIAL LICENSE AGREEMENT RESTRICTED RIGHTS LEGEND Use, duplication, or disclosure by the Government is subject to restrictions as set forth in subparagraph. (c) (1) (11) of the rights in Technical Data and computer software clause of DFARS 52.227-7013. Hitachi Software Engineering America, Ltd. (HISAL) 601 Gateway Boulevard, Suite 100 South San Francisco, CA 94080 GENERAL This agreement will be governed by the laws of the State of California, except for that body of law dealing with conflicts of law. Future updates of the Software will be available for purchase by licensees for a fee provided a registration card has been received by Hitachi Software Engineering America, Ltd. Should you have any questions concerning this Agreement, you may contact HISAL at http://www.miraibio.com. You acknowledge that you have read this Agreement, understand it and agree to be bound by its terms and conditions. You further agree that it is the complete and exclusive statement of the agreement between us which supersedes any proposal or prior agreement, oral or written, and any other communications between us in relation to the subject matter of this Agreement. MasterPlex ReaderFit www.miraibio.com 2
CONTENTS 1 Welcome... 5 1.1 About This Tutorial... 5 1.2 Technical Support... 6 2 Analysis Process... 7 2.1 Analysis flow... 7 3 Importing data... 8 3.1 Opening files from the application icon... 8 3.2 Opening files from the File menu... 9 3.3 File Importing... 9 4 Configuring Plate Layout... 14 4.1 Well marking... 14 4.2 Background samples... 15 4.3 Standard samples... 16 4.4 Control samples... 17 4.5 Unknown samples... 18 4.6 Unmarking wells... 19 4.7 Auto-Drag Replicate Grouping... 20 5 Standard Curves... 23 5.1 Inputting Standard Values... 23 5.2 Standard Replicates... 25 5.3 Inputting dilution factors for unknowns and controls... 26 5.3 Inputting dilution factors for unknowns and controls... 27 5.4 Choosing a standard model equation... 29 5.5 Calculation... 31 5.6 Best fit... 32 6 Reviewing data... 33 6.1 Data table... 33 6.2 Arranging Data columns... 34 6.3 Generating Charts... 35 7 Templates... 38 7.1 Template Manager... 38 8 Other features... 40 8.1 Export Manager... 40 8.2 Virtual Plates... 42 MasterPlex ReaderFit www.miraibio.com 3
8.3 Copy and Paste sample name... 45 MasterPlex ReaderFit www.miraibio.com 4
CHA PT E R 1 WELCOME CHAPTER 1 Welcome Welcome to the MasterPlex ReaderFit tutorial. MasterPlex ReaderFit software analyzes results files (*.csv, *txt or *.xls) from various plate reader instruments. 1.1 About This Tutorial This tutorial explains how to use the MasterPlex ReaderFit software to: import results files (*.csv, *.txt or *.xls) from plate reader designate standard, unknown, control, and background wells generate standard curves compute analyte concentrations review data in data tables and generate data charts What s new in MasterPlex ReaderFit MasterPlex ReaderFit offers several new features: New and Improved Curve Fitting Best Fit feature automatically selects the optimal model equation and weighting algorithm On the fly recalculations for easy outlier exclusion Brand New Report Generator Spreadsheet style pivot charts Drag and drop to create your own custom report Completely Redesigned Tabbed UI Simple left to right workflow Context related items grouped in tabs Brand New Graphing Engine Create customized publication quality charts and graphs with a few clicks Redesigned Plate View Display multiple data points per well View statistical data for replicate groups at a glance MasterPlex ReaderFit www.miraibio.com 5
CHA PT E R 1 WELCOME 1.2 Technical Support You can contact MiraiBio Technical support at: Hitachi Software Engineering America, Ltd. 601 Gateway Boulevard, Suite 100 South San Francisco, CA 94080 USA Tel: +1 (650) 615-7600 Toll Free: +1 (888) 615-9600 Fax: +1 (650) 615-7639 E-mail: support@miraibio.com www.miraibio.com MasterPlex ReaderFit www.miraibio.com 6
CHA PT E R 2 ANALYSIS PROCESS CHAPTER 2 Analysis Process 2.1 Analysis flow The following flowchart shows typical absolute quantification analysis. In this tutorial you will learn how to generate calculated data and review the data according to this flow. Import file Assign analytes in the plate Configure plate layout (Mark standard, unknown, control and background samples) Input standard values Choose a model equation and set calculation options Generate standard curves and calculate unknown concentrations Review data on data table or chart, print or save data MasterPlex ReaderFit www.miraibio.com 7
CHA PT E R 3 IMPORTING DATA CHAPTER 3 Importing data 3.1 Opening files from the application icon Step1 Click MasterPlex ReaderFit icon in the Applications pane. Step2 Select an output file from a plate reader instrument. MasterPlex ReaderFit can open the following types of file. File extension Description 1.csv Comma separated valus 2.txt Text file 3.xls Excel file MasterPlex ReaderFit www.miraibio.com 8
CHA PT E R 3 IMPORTING DATA 3.2 Opening files from the File menu You can also open output files from File menu. 3.3 File Importing MasterPlex ReaderFit has a flexible file importer wizard which enables you to import your plate reader result data manually. Step1 Open result file from plate reader. File import wizard page appears. If your result data is already patterned in the ReaderFit program, wizard takes you to step5 automatically. MasterPlex ReaderFit www.miraibio.com 9
CHA PT E R 3 IMPORTING DATA Step2 Select one of the deliminator type from select deliminator type box so that your result data is correctly delimited in the file display grid. Step3 Select data format type from select data format box, plate or list. Step4 Select data area you want to import by mouse dragging. MasterPlex ReaderFit www.miraibio.com 10
CHA PT E R 3 IMPORTING DATA Step5 Click next to proceed, then Analyte Assignment page appears Analyte mode Mini Plate view Analyte list (If there is only one analyte in the plate) Step6 Make sure the Single Analyte radio button is selected, then click Finish button at the bottom of the dialog. MasterPlex ReaderFit www.miraibio.com 11
CHA PT E R 3 IMPORTING DATA (If there are more than one analyte in the plate) Step6 Select the Multiple analytes radio button. Step7 Highlight the cells you want to assign as one analyte by mouse dragging or pressing CTRL key. Step8 Click Assign button, then the. wells are filled certain color and the analyte with default name is added in the analyte list. MasterPlex ReaderFit www.miraibio.com 12
CHA PT E R 3 IMPORTING DATA Step9 Change the analyte name or color. Then repeat analyte assignment until all blank wells have been assigned. When all wells were assigned, click finish to open the plate view. MasterPlex ReaderFit www.miraibio.com 13
CHA PT E R 4 CONFIGURING PLATE LAYOUT CHAPTER 4 Configuring Plate Layout Before proceeding to the concentration calculation you must indicate sample types in the plate view. 4.1 Well marking The first thing you have to do is to mark wells as certain sample types. Plate view Background sample Standard sample Unknown sample MasterPlex ReaderFit www.miraibio.com 14
CHA PT E R 4 CONFIGURING PLATE LAYOUT 4.2 Background samples Step1 Select background well(s) in the plate view. Step2 Click the background (B) icon located at the top of the plate view. Alternately you can right click on the plate view, then select background (B). Step3 Background sample wells are now colored brown and automatically grouped. The black border denotes a replicate group. - Note - If you want to subtract background values from all RLU values, please make sure the background subtraction feature is enabled. Default is ON. MasterPlex ReaderFit www.miraibio.com 15
CHA PT E R 4 CONFIGURING PLATE LAYOUT 4.3 Standard samples Step1 Select standard wells in the plate view. Step2 Click the standard (S) icon located at the top of the plate view. Alternately you can right click on the plate view, then select standard (S). Step3 Standard sample wells are now colored blue. MasterPlex ReaderFit www.miraibio.com 16
CHA PT E R 4 CONFIGURING PLATE LAYOUT 4.4 Control samples Step1 Select the control well(s) in a given replicate group. Step2 Click the control (C) icon located at the top of the plate view. Alternately you can right click on the plate view, then select control (C). Repeat Step 2 for each unknown replicate group. Hint: If your replicate groups are laid out similarly across your plate you can use the Auto-drag feature in MasterPlex to copy the replicate grouping pattern across the rest of the plate. See below. Step3 Control sample wells are now colored yellow and automatically grouped. The black border denotes a replicate group. MasterPlex ReaderFit www.miraibio.com 17
CHA PT E R 4 CONFIGURING PLATE LAYOUT 4.5 Unknown samples Step1 Select the unknown well(s) in a given replicate group. Step2 Click the unknown (U) icon located at the top of the plate view. Alternately you can right click on the plate view, then select unknown (U). Repeat Step 2 for each unknown replicate group. Hint: If your replicate groups are laid out similarly across your plate you can use the Auto-drag feature in MasterPlex to copy the replicate grouping pattern across the rest of the plate. See below. Step3 Unknown sample wells are now colored green and automatically grouped. The black border denotes a replicate group. MasterPlex ReaderFit www.miraibio.com 18
CHA PT E R 4 CONFIGURING PLATE LAYOUT 4.6 Unmarking wells Step1 Select the well(s) you would like to unmark. Step2 Click the unmark (X) icon located at the top of the plate view. Alternately you can right click on the plate view, then select unmark (X). -Note- If you unmark standard wells you will be notified that no standard groups are currently marked. MasterPlex ReaderFit www.miraibio.com 19
CHA PT E R 4 CONFIGURING PLATE LAYOUT 4.7 Auto-Drag Replicate Grouping If the unknown or control replicate groups are laid out similarly on the plate you can use the auto-drag feature to quickly copy the replicate pattern across all relevant wells. Step1 Mark one of the unknown or control replicate groups and select all the wells in the group. (The group border turns from black to red color.) ( Select all feature) While hovering over a replicate group border the mouse cursor changes to a hand icon. If you click the group border with the hand icon all wells in the group are selected. MasterPlex ReaderFit www.miraibio.com 20
CHA PT E R 4 CONFIGURING PLATE LAYOUT Step2 After the group is selected you will see a small black square mark on the bottom right of the group. While hovering over this square the cursor turns to a cross mark. Step3 While holding the left mouse button move the mouse to the right. Step4 Then move the mouse down. MasterPlex ReaderFit www.miraibio.com 21
CHA PT E R 4 CONFIGURING PLATE LAYOUT (Vertical auto-drag replicate grouping) Move the mouse down first. Then move the mouse right. MasterPlex ReaderFit www.miraibio.com 22
CHA PT E R 5 STANDARD CURVES CHAPTER 5 Standard Curves Before generating standard curves you need to input known standard values. 5.1 Inputting Standard Values (Manual input) Step1 Change the data display to Standards Standard values are displayed. Step2 Select one of the standard wells (well turns gray). Then click the same well again. You are now in edit mode. Input the desired value and click another well or press enter to record the change. MasterPlex ReaderFit www.miraibio.com 23
CHA PT E R 5 STANDARD CURVES (Auto fill) Step1 Click the Autofill icon located at the top of the plate view. The Auto Fill dialog pops up. Step2 1) Select an analyte or check Fill for all analyte sets. 2) Select the Standard group if more than one Standard group has been marked. 3) Input the starting concentration. 1 2 3 4 5 4) Input the dilution factor. 6 5) Choose the replicate number and orientation. You can see the various directions in the details window. 6) Choose dilution direction. You can see the various directions in details window. 7 7) Press the Fill & Close button If you want to continue to input standard values press Apply and select the next analyte. MasterPlex ReaderFit www.miraibio.com 24
CHA PT E R 5 STANDARD CURVES 5.2 Standard Replicates If the standard replicates are pipetted in a side by side orientation select None in the Replicate Numbers drop down menu. The software will automatically fill in standard values for all replicate groups. MasterPlex ReaderFit www.miraibio.com 25
CHA PT E R 5 STANDARD CURVES If the standard replicates are pipetted in a stacked orientation select the number of replicates in Replicate Numbers drop down menu. The software will automatically fill in standard values for all replicate groups. MasterPlex ReaderFit www.miraibio.com 26
CHA PT E R 5 STANDARD CURVES 5.3 Inputting dilution factors for unknowns and controls Step1 Change the data display to Dilution Step2 Select a well and enter edit mode. Input desired value and hit enter. MasterPlex ReaderFit www.miraibio.com 27
CHA PT E R 5 STANDARD CURVES (Batch input) Step1 Change the data display to Dilution Step2 Select multiple wells. Step3 Right click on the selected wells and click Plate Dilution. Step4 The plate dilution dialog pops up. Enter desired value and click OK. Step5 Values are changed for all selected wells. MasterPlex ReaderFit www.miraibio.com 28
CHA PT E R 5 STANDARD CURVES 5.4 Choosing a standard model equation Step1 Click the Standard Curve tab. MasterPlex ReaderFit www.miraibio.com 29
CHA PT E R 5 STANDARD CURVES Step2 1) Select the model equation you want to apply to your standard data. 2) Check Select for all analytes box if you want to apply this equation for all analytes. 3) Choose a weighting option. 4) Choose data points display option. 1 2 3 4 MasterPlex ReaderFit www.miraibio.com 30
CHA PT E R 5 STANDARD CURVES 5.5 Calculation Click the button. MasterPlex ReaderFit will generate the standard curve and calculate concentrations for all unknown samples. MasterPlex ReaderFit www.miraibio.com 31
CHA PT E R 5 STANDARD CURVES 5.6 Best fit MasterPlex ReaderFit has an auto calculation button named BEST FIT. This button is located at the top of the plate view in Plate tab. Click this button after configuring your plate and inputting your standard values. MasterPlex ReaderFit will automatically choose the best equation and weighting combination, calculate unknown concentrations and switch to the standard curve tab. Click BEST FIT ReaderFit begins searching for the best combination After searching, the standard curve tab displays automatically. MasterPlex ReaderFit www.miraibio.com 32
CHA PT E R 6 REVIEWING DATA CHAPTER 6 Reviewing data 6.1 Data table To review the calculated data, click the "Data Table" tab. By default, data is displayed in columns and sorted by analyte. MasterPlex ReaderFit www.miraibio.com 33
CHA PT E R 6 REVIEWING DATA 6.2 Arranging Data columns Step1 Click the Show Columns Selector button, located at the top of the data table. Step2 The Column selector will pop up and you will see various data types which can be displayed in the data table. Step3 Drag one of the data types to the column bar in the data table Step4 The new data type will be added as a column to the report. MasterPlex ReaderFit www.miraibio.com 34
CHA PT E R 6 REVIEWING DATA 6.3 Generating Charts To generate a chart click the Chart tab. Step1 Select the data type for y-axis Value and select chart type for the x-axis data series. Step2 Click the well selector icon on the top of the view, then select wells to display in the chart. You can select multiple wells by dragging the mouse or by clicking while holding down the [CTRL] key. MasterPlex ReaderFit www.miraibio.com 35
CHA PT E R 6 REVIEWING DATA Step3 Choose one of the chart styles by clicking the down arrow gallery icon. on the chart MasterPlex ReaderFit www.miraibio.com 36
CHA PT E R 6 REVIEWING DATA You can easily customize your chart on the chart properties. (click ) Example: 3D Manhattan bar style MasterPlex ReaderFit www.miraibio.com 37
CHA PT E R 7 TEMPLATES CHAPTER 7 Templates MasterPlex ReaderFit has a useful plate template feature. You can apply the same plate configuration for your routine experiments. 7.1 Template Manager Step1 To open the template manager, click the template icon located at the top of the plate view in the plate tab. Step2 Click the Save button in the dialog box. Step3 Input the template name and description(optional) for the template. MasterPlex ReaderFit www.miraibio.com 38
CHA PT E R 7 TEMPLATES Step4 Saved templates are shown in the available templates list. When you want to apply one of the templates to the current plate, select the template in the list, then click Load button. MasterPlex ReaderFit www.miraibio.com 39
CHA PT E R 8 OTHER FEATURES CHAPTER 8 Other features MasterPlex ReaderFit has many other features. In this chapter, it guides you how to use export manger and virtual plate. 8.1 Export Manager Export Manager is an advanced feature for data exporting. You can export the data in a custom data format by using XSL style sheets. Step1 To use the advanced export feature, go to the Export Manager tab. Step2 Choose one of the style sheets listed in the Style Sheet window, then click apply button. MasterPlex ReaderFit www.miraibio.com 40
CHA PT E R 8 OTHER FEATURES Step3 The saving dialog for transformed data is displayed. Input file name, then click save button. The transformed data is displayed in the data viewer if the transformed data format is html, txt or csv. You can print transformed data from the menu on the viewer. - Note - For more details about Style Sheet, please contact our support. We are also offering making your desired output by several kinds of file formats (html, csv, xls or xml). MasterPlex ReaderFit www.miraibio.com 41
CHA PT E R 8 OTHER FEATURES 8.2 Virtual Plates A virtual plate is a software simulation of an empty microtiter plate. User-selected unknown and standard data from one or more actual plates (.csv,.txt,.xls) can be added to a virtual plate. In a virtual plate you can: Compare and analyze samples from user-selected actual plates (.csv,.txt,.xls or.mxqs) Specify custom row and column dimensions to increase analysis throughput Generate a Dose-Response curve for user-selected data Step1 To generate a virtual plate, click virtual plate icon located on the application menu bar. Step2 Select application type, then input row and column size of the virtual plate you want to generate. MasterPlex ReaderFit www.miraibio.com 42
CHA PT E R 8 OTHER FEATURES Step3 An empty virtual plate is shown in the plate view. Step4 Go to the original data plate view and select the well(s) you want to copy to the virtual plate. Then right click on the well(s) and choose Aspirate. Step5 Go back to virtual plate and select the top left well of the area you want to paste the data and select Dispense. MasterPlex ReaderFit www.miraibio.com 43
CHA PT E R 8 OTHER FEATURES Step6 Now you will see the copied data on the virtual plate. You can copy more data from other plates, and mark samples and calculate on the virtual plate the same as a normal plate. MasterPlex ReaderFit www.miraibio.com 44
CHA PT E R 8 OTHER FEATURES 8.3 Copy and Paste sample name You can copy the sample name from plate view or from various platform. Also you can paste the copied text as sample name on the plate view. Step1 Copy text block for the sample name from text file or xls file. Step2 Select the top left cell you want to paste the text block as sample name Step3 Right click and select Paste sample name MasterPlex ReaderFit www.miraibio.com 45
CHA PT E R 8 OTHER FEATURES (Copy from/paste to situations) Text rows Microsoft Excel cells Tab delimited text block Comma delimited text block MasterPlex ReaderFit www.miraibio.com 46