Thermal Cycler Dice TM Real Time System Software. Operation Manual

Transcription

1 Thermal Cycler Dice TM Real Time System Software Version 4.02 Code TP900/TP960 Operation Manual Attention: Thermal Cycler Dice TM Real Time System Software Operation Manual should be stored for reference in a secure and accessible location.

2 This document applies to the control and analysis software of the multi-wavelength model Thermal Cycler Dice TM Real Time System. <Multi-wavelength models> Thermal Cycler Dice TM Real Time System II (Cat.# TP900) Thermal Cycler Dice TM Real Time System II MRQ (Cat.# TP860) * For hardware use, refer to the separate user's manual "Hardware Maintenance." * For use of Multiplate RQ software for multi-plate relative quantification, refer to the separate Multiplate RQ user's manual. NOTICE P10 PCR Notice (For outside US) Purchase of this instrument conveys a limited non-transferable immunity from suit for the purchaser's own internal research and development and applied fields other than human in vitro diagnostics under one or more of U.S. Patents No. 5,038,852, 5,656,493, 5,333,675, 5,475,610, and 6,703,236 (claims 1-6 only), or corresponding claims in their non-u.s. counterparts, owned by Applera Corporation. No right is conveyed expressly, by implication or by estoppel under any other patent claim. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Applied Biosystems does not guarantee the performance of this instrument. L45 Heated Cover This product is covered by the claims of U.S. Patent 5,552,580, and their corresponding foreign counterpart patent claims. L47 Real-Time PCR Quantification Method The purchase of this product includes a limited, non-transferable license for all fields other than human or veterinary in vitro diagnostics under specific claims of U.S. Patent Nos. 6,174,670, 6,569,627, 6,303,305, and 6,503,720, owned by the University of Utah Research Foundation and licensed to Idaho Technology, Inc. and Roche Diagnostics GmbH.

3 CONTENTS 1. Main Parts of the System Software Overview 2-1. Operation Flow Startup and Shutdown (1) Startup... 8 (2) Shutdown Layout and Operation of Each Main Screen (1) Initial Screen... 9 (2) Experiment Options Screen (3) Plate Setup Screen (4) Thermal Profile Setup Screen (5) Result/Analysis Screen (6) Output Setup Screen (7) Basic Tools Initial Settings (1) Registering or deleting a user (2) User Setting (Modifying default settings) (3) Filter Settings File Operation (1) Creating a New Experiment File (2) Using an Existing Experiment File (3) Experiment File Management Performing a Run 3-1. Experiment File Settings (1) Creating a New Experiment File (2) Changing the Experiment Type Set up the PCR Master Mix (1) Preparation of PCR Master Mix (2) Setting up the Plate/Tube Setting the PCR Conditions (1) Basic Settings (2) Advanced Settings

4 3-4. Run (1) Starting a run (2) Monitoring Run Status (3) Instrument Control during a Run (4) Real Time Results Display (5) Run Completion (6) Run History Verification Basic Operations 4-1. Plate Document Settings (1) Basic Operations (2) Convenient functions Basic Operations of Analysis Changing the Graph Properties Absolute Quantification 5-1. Plate Document Settings Analysis (1) Analysis Parameter Settings (2) Analysis Results Display (3) Text Report Relative Quantification 6-1. Plate Document Settings Analysis (1) Analysis Parameter Settings (2) Analysis Results Display (3) Text Report Plus/Minus Assay 7-1. Plate Document Settings Analysis (1) Analysis Parameter Settings (2) Analysis Results Display (3) Text Report

5 8. SNP Genotyping Assay 8-1. Plate Document Settings Analysis (1) Analysis Parameter Settings (2) Analysis Results Display (3) Text Report Exporting and Printing 9-1. Exporting (1) Plate Document and Thermal Profile (2) Graphs (3) Numerical Data (4) Reports Printing (1) Plate Document and Thermal Profile (2) Graphs (3) Reports Exporting Files for RDML Hardware Settings Halogen Lamp (1) Turning the Lamp ON/OFF (2) Lamp Status Verification and Reset after Replacement Appendix Glossary Troubleshooting References

6 1. Main Parts of the System [Lid Closed] Ventilation Intake (for cooling halogen lamp) LED Display Area Lid Handle Ventilation Intake (front side) Power Switch ON ( ), OFF (O) Ventilation Intake (left and right sides) LED Display Area POWER STANDBY RUN Illuminated when the power switch is in the ON ( ) position. Dark when the power switch is in the OFF (O) position. Blinks when the halogen lamp and lid are warming up. Illuminated when the halogen lamp and lid have finished warming up. Dark when the equipment is performing a run. Illuminated when a run is being performed. 1. Main parts of the system 4

7 [Lid Open] LED Display Area Lid Lock Catch Ventilation Intake (for cooling halogen lamp) Lid Handle Lid Sample Block Ventilation Intake (front side) Power Switch ON ( ), OFF (O) Ventilation Intake (left and right sides) 5

8 [Back] Top Cover Power Cable Connector Ventilation exhaust (back) USB Cable Connector 1. Main parts of the system 6

9 2. Software Overview 2-1. Operation Flow System Startup 1) Start Up the Thermal Cycler Dice Real Time System II Main Unit Turn the Power Switch at the bottom right of the front panel to ON. 2) Start up Computer 3) Launch the Thermal Cycler Dice Real Time System Software Double-click the shortcut icon on the desktop to launch the software. Setup of Experiment Files and the Reaction Plate/Tube 4) Create a New Experiment File Select New from the File menu and set the Experiment Type and User ID. 5) Prepare the PCR Master Mix 6) Set the Plate/Tube Lift the handle of the main unit and set the plate or tube on the sample block. Setup of PCR Conditions and Starting a Run 7) Set up the PCR Conditions Set up the PCR conditions in the Thermal Profile Setup screen. 8) Perform a Run Click the Start Run button in the Thermal Profile Setup screen. 9) Real Time Monitoring of the Fluorescence Data Fluorescence data is displayed in the Result/Analysis screen in real time. Sample Information setting and Data Analysis 10) Set up Plate Document Enter the sample information in the Plate Setup screen. 11) Data Analysis Set the baseline, threshold level, and other parameters and perform analysis. 12) Analysis Results Output System Shutdown 13) Software Termination 14) Computer Shutdown 15) Shutdown of the Main Unit Turn the Power Switch at the bottom right of the front panel to OFF. 7

10 2-2. Startup and Shutdown (1)Startup a. Verify that the Thermal Cycler Dice Real Time System II main unit is connected to the computer by the USB cable. b. Turn the power switch at the lower right of the front panel to the ON ( ) position. [Note] Turning the power ON for the main unit turns the halogen lamp on at the same time. Allow the machine to warm up for about 15 minutes, until the intensity of the lamp light stabilizes. Then allow the lid heater to warm up until the temperature is greater than 105ºC. The STANDBY indicator blinks while the machine is warming up. When both the halogen lamp and the lid heater have finished warming up, the indicator is steadily illuminated, and the equipment may then be used. c. Next, turn the computer power ON. d. Once the computer has completed its start up sequence, double-click the Thermal Cycler Dice Real Time System software icon on the desktop. [Note] The software can also be launched by clicking on the start menu at the lower left of the screen and selecting [Program] > [TaKaRa Thermal Cycler Dice] > [Thermal Cycler Dice Real Time System Software]. e. The splash screen shown at right is displayed, and the software launches. f. The software initial screen is displayed. [Note] The connection status can be verified by checking instrument status (right port) in the status bar at the bottom of the screen. If properly connected, then Connected will be displayed. If not connected, or if the main unit power is OFF, then Disconnected is displayed. (2) Shutdown a. Select Exit from the File menu of the software to terminate the software. b. Select Turn off Computer from the start menu at the bottom left of the screen. c. The shutdown screen is displayed. d. Click Turn Off to shut down the computer. e. Turn the power switch of the Thermal Cycler Dice Real Time System II main unit to the OFF (O) position. 2. Software Overview 8

11 2-3. Layout and Operation of Each Main Screen (1) Initial Screen The initial screen is displayed when the Thermal Cycler Dice Real Time System software is launched. This is where the initial software options are set. [Initial Screen Display Method] o o The Initial Screen is displayed when the Thermal Cycler Dice Real Time System software is launched. When the Thermal Cycler Dice Real Time System software is in use, closing all open experiment files brings the system back to the initial screen. [Main Operations Performed from the Initial Screen] o o o o o o o Create new experiment files. Open existing experiment files. Manage experiment files with the Data Manager. Verify the ON/OFF status and the hours of usage of the halogen lamp in the main unit, or reset the remaining hours of usage after a bulb has been replaced. Optically calibrate using calibration plate. Carry out the thermal cycler (main equipment) self-diagnostic function. Verify the filter set. 9

12 (2) Experiment Options Screen Set the Experiment Type and User ID in this screen. Description is a free space you can note experimental information etc. [Experiment Options Screen Display Method] o Click the Experimental Options button in the side area, or select [Section] > [Experiment Options] from the menu bar. [Main Operations Performed from the Experiment Options Screen] o o o Setting and verification of Experiment Information The Experiment Information is displayed and you can change it, if needed. Description is a free space you can note experimental information etc. Setting and verification of User Information The User Information is displayed and you can change it, if needed. You can also add a new user or change the information of existing user by clicking the Edit button. In addition to the above, almost all operations that can be performed from the initial screen can also be performed from this screen. 2. Software Overview 10

13 (3) Plate Setup Screen Set the sample information in this screen. First, make a Target List and Sample List, and then set Target and Sample for each well on the Plate Image. [Plate Setup Screen Display Method] o o The Plate Setup screen is displayed when a new experiment file is created or when an existing experiment file is opened. If switching from another screen, click the Plate Setup button in the area on the left of the screen, or select [Section] > [Plate Setup] from the menu bar. [Main Operations Performed from the Plate Setup Screen] o o o o Make the Target List and Sample List Set the information of Targets and Samples in the Target&Sample Setting screen. Make the Plate Image Set Target and Sample for each well on the Plate Image. Save and print the Plate Setup screen as a figure or in report format. In addition to the above, almost all operations that can be performed from the initial screen can also be performed from this screen. 11

14 (4)Thermal Profile Setup Screen The Thermal Profile Setup screen is for setting the PCR conditions and selecting the fluorescence filter for measuring. It is also possible to start a run from this screen. [Thermal Profile Setup Screen Display Method] o Click the Thermal Profile Setup button in the side area, or select [Section] > [Thermal Profile Setup] from the menu bar. [Main Operations Performed from the Thermal Profile Setup Screen] o o o o o o Set PCR conditions. Select the fluorescence filter used for measuring. Start a run and control instrument during a run. A run in progress can also be paused (and restarted) or forcibly terminated. Monitor the progress and status of a run. Save and print the Thermal Profile screen as a figure or in report format. In addition to the above, almost all operations that can be performed from the initial screen can also be performed from this screen. 2. Software Overview 12

15 (5) Result/Analysis Screen Using the Result/Analysis screen, data for the current run can be monitored in real time. After completion of the run, the measured data is analyzed and the results are displayed. [Result/Analysis Screen Display Method] o Click the Result/Analysis button in the side area, or select [Section] > [Result/Analysis] from the menu bar. [Main Operations Performed from the Result/Analysis Screen] o o o o Display real time data during the run. Set analysis parameters and perform analysis after the run is executed. o Display Amplification Plots and calculate Ct values (Threshold Cycle). o Display the Standard Curve and quantify unknown samples. o Calculate the Relative Quantification value and display the graph (if Relative Quantification is used). o Display the Dissociation Curve and calculate Tm values. Save and print the measured values, analysis results, analysis settings etc. in graph or report format. In addition to the above, almost all operations that can be performed from the initial screen can also be performed from this screen. 13

16 (6) Output Setup Screen Select files you need for making of RDML file, and export them. * This screen is only for Absolute Quantification and Relative Quantification. [Output Setup Screen Display Method] o Click the Output Setup button in the side area, or select [Section] > [Output Setup] from the menu bar. [Main Operations Performed from the Output Setup Screen] o o o Select files you need for making of RDML file. Export files for RDML. In addition to the above, almost all operations that can be performed from the initial screen can also be performed from this screen. 2. Software Overview 14

17 (7) Basic Tools The basic tools are commonly used tools available on each screen, including the menu bar, the toolbar, the status bar, and the side area. Menu Bar, Tool Bar Side Area Status Bar [Menu Bar] Every command can be performed by making a selection from a menu. Menu Bar Contents File New Create a new experiment file Open Close Data Manager Open an existing experiment file Close an experiment file that is open Display the Data Manager Screen for experiment file Save Save by overwriting the existing experiment file Save As Save by assigning a user-defined name Load Template Load an existing file as a template Make Full Report Output a report and exports data and graphs Print Print report Exit Terminate the Thermal Cycler Dice Real Time Software Edit Copy Copy the contents of the selected area Paste Paste copied contents Undo Cancel the previous operation Redo Re-perform an action that has been cancelled by Undo Section Experiment Options Display the Experiment Options Screen Plate Setup Display the Plate Setup Screen Thermal Setup Display the Thermal Profile Setup Screen Result/Analysis Display the Result/Analysis Screen Output Setup Display the Output Setup Screen Analysis Data Profile Display the Data Profile window Analysis Data Selection Smoothing Display the Analysis Data Selection window Display the Smoothing Setting window Instrument Run Start Start a run Add Cycle Add cycles during a run Pause Pause a run in progress 15

18 Restart Restart a run that is paused Skip to Next Skip to the next Pattern Stop Forcibly terminate a run Lamp On Turn ON the Halogen Lamp of the main unit Lamp Off Turn OFF the Halogen Lamp of the main unit Lamp Status Display Halogen Lamp usage time and reset timer after lamp has been replaced Filter Set Master Data Filter information settings and additional filter settings Localized Set Filter information for the file under analysis Calibrate Optical Display the Optical Calibration Screen Thermal Maintenance Mode (not used) Diagnosis Self-diagnostic function for Thermal Cycler Dice Real Time main unit User Change Change a user Setting Management Display the User Setting screen Display the User Management screen Window Cascade Display multiple experiment files in cascade view Tile Horizontal Display multiple experiment files tiled horizontally Tile Vertical Display multiple experiment files tiled vertically < Experiment File > Switch the display between experiment files Help Help Display Help About [Toolbar] Commands are executed by clicking on the icons. Display version Information a b c d e f g h i j k l a New Experiment Creates a new experiment file b Open Experiment Opens an existing experiment file c Save Experiment Specifies a name to a new experiment file and saves it, or overwrites the existing file d Print Experiment Prints a report after setting options on the Report Setting Screen e Copy Copies the contents of the selected area f Paste Pastes the copied contents g Undo Cancels the previous operation h Redo Returns to the state pertaining before the Undo operation was performed i Zoom In Enlarges the Results/Analysis Screen graph selection area j Zoom Out Returns to the state before enlargement via the Zoom In operation k Lamp On/Off Shows whether the Halogen Lamp is ON/OFF l Help Displays Help [Status Bar] The Status Bar displays the following information. a b c d e 2. Software Overview 16

19 a Run Status Displays the filename of the run in progress b Lamp Status Lamp Total Time Displays the state of the Halogen Lamp of the main unit (ON/OFF) Displays the hours of usage of the Halogen Lamp in the main unit c Lid Status Lid Displays the status of the main unit Lid (Open/Close) Temperature Displays the temperature of the Lid (Units: ºC) d Instrument Status Displays the connection status of the PC to the main unit (Connected/Disconnected) e Camera Status Displays the connection status of the PC to the camera (Connected/Disconnected) [Side Area] The side area displays the following buttons and information. Options g~j are displayed when a run is in progress. a b c d e f g h i j Constant Display Run in Progress a Experiment Options Experiment Options Screen display b Plate Setup Plate Setup Screen display c Thermal Profile Setup Thermal Profile Setup Screen display d Result/Analysis Result/Analysis Screen display e Output Setup Output Setup Screen display f Information (Plate Setup Screen only) Samples of well information g Time/Temperature Remaining Time Remaining time until run completion Lid Temperature Block Temperature Current Lid temperature display Current Block temperature display h Running Point Pattern Number of the pattern in progress Segment Cycle Number of the segment in progress Number of the cycle in progress i Instrument Control Add Cycle Adds a cycles during a run Pause Restart Skip to Next Stop Pauses a run Restarts a paused run Skips to next pattern Forcibly terminates a run j Turn lamp off after run When checked, the Halogen Lamp is turned OFF after the run. 17

20 2-4. Initial Settings (1) Registering or deleting a user Thermal Cycler Dice TM Real-Time System users can be registered or deleted. User registration is optional, but it s useful for managing the experiment files and you can change the default settings of analysis, graph display, etc. for each users. [Registration of User] Register a Thermal Cycler Dice TM Real-Time System user as follows: a. Select Management from the User menu to display the User Management screen. b. Click the New button to creat a new user. c. After entering user information, click the Add button. d. After confirming that the new user is displayed on the list, click the OK button. [Deletion of User] a. Select Management from the User menu to display the User Management screen. b. Select a user to delete from the list, and click the Delete button. c. After confirming that the user is not displayed on the list, click the OK button. [Select/Chang a User] [Select a user] Select a user when you creat an experiment file. a. Select New from the File menu to display the New Experiment Options screen. b. From the User ID list, select a user and click the OK button. [Chang a user] Change the user of existing experiment file. a. Select Change from the User menu. b. Select a user you want to change to. 2. Software Overview 18

21 (2) User Setting (Modifying default settings) Various default settings (analytical parameters, graph display method, etc.) can be modified and saved for each user. This is done on the User Setting screen. [Displaying the User Setting screen] Display the User Setting screen by either of the following methods: [From the New Experiment Options screen] a. Select New from the File menu to display the New Experiment Options screen. b. Click the Edit button on the New Experiment Options screen to display the User Management screen. c. Double-click a user for selection. [From the Management menu] a. Select Management from the User menu to display the User Management screen. b. Double-click a user for selection. Double-click [From an experiment file] a. Select the experiment file where the user is set. b. Select Settings from the User menu. 19

22 [Modifying and saving default settings] The User Setting screen has six tabs. Change screens by clicking these tabs. After modifying settings, click the OK button to update the settings. If the Cancel button is clicked, the settings are not updated. Clicking the Reset button restores the default settings of the software. [User Information] Set user information on this screen. For details about each item, see 2-4 (1). [Analysis] Set analytical items on this screen. For details about each item, see 5-2 (1) and 7-2 (1). [Full Report/Print] Set report output and printing items on this screen. For details about each item, see 9-1 (4). 2. Software Overview 20

23 [Chart Properties] Set graph display items on this screen. For details about each item, see 4-3. [Text Report] Set text report display items on this screen. For details about each item, see 5-2 (3), 6-2 (3), 7-2 (3), and 8-2 (3) [Lamp] Set "Turn lamp off after run" on this screen. This setting becomes valid from the next setting. For details about this function, see 3-4 (3). 21

24 (3) Filter Settings Two types of filter are installed as standard in the Thermal Cycler Dice Real Time System II : FAM /SYBR Green I, and ROX /Texas Red. Two additional types of filter can be installed as an option. The setup method for each of these filters is explained in this document. [Master Data Settings] The master data settings are used to set the type of fluorescent dye to be measured for each filter. The master data settings are applied to all experiment files. a. Make sure the initial screen is displayed. b. Select [Instrument] > [Filter Set] > [Master] from the menu bar. The Filter Setting window is displayed. If using operation system Windows Vista or Windows 7, click YES on the UAC message. As the display is closed and opened, re-select same steps again. c. Select the filter to which the setting changes are to be applied and double-click to display the Filter Editor. d. Select the name of the fluorescent dye from the Dye Name drop-down list. Filter Name: Name of the filter (fixed). Dye Name: Name of the fluorescent dye used for detection (can be selected from list). e. Optional colors can be selected by clicking Display Color. f. To change the settings of a different filter, click the Prev or Next button. g. Click OK. The altered filter information is displayed in Filter Info. [Localized Data Settings] The localized data settings are the same as the master data settings, but the localized data settings are only applied to the experiment file currently under analysis. a. With the experiment file open, select [Instrument] > [Filter Set] > [Localized Data] from the menu bar and perform the operations as described above. [Note] Click a tab of Filter Setting to switch the display. Normally, it is not necessary to change the Position & Order and Factor settings. The Position & Order tab is not displayed for the Localized Data Settings. Filter Info: Displays information related to filters and settings. Position & Order: Displays filter position and measuring order settings. Factor: Settings for the factors of additional filters. 2. Software Overview 22

25 2-5. File Operation (1) Creating a New Experiment File Create a new experiment file to perform a run. Select [File] > [New] from the menu bar. The New Experiment Options window is displayed. After setting experiment type and user ID, click the OK button. A new experiment file is opened. (Refer to section 3-1.) (2) Using an Existing Experiment File It is possible to open and operate multiple files at the same time. While a run is in progress for one file, a different file may also be open for changing file settings and data analysis. [Opening a File] o Select [File] > [Open] from the menu bar or click the Open icon in the toolbar. Select the file to be opened in the Open window. o Select [File] > [Data Manager] from the menu bar. The Data Manager window is displayed. Select a file and click the Open button. o The file can also be opened by double-clicking on its file icon, by dragging and dropping the file icon into the Thermal Cycle Dice Real Time System software screen, or by dragging and dropping the file icon onto the Thermal Cycle Dice Real Time System software icon. [Switching Between Multiple Files and Simultaneous Display] o Selecting [Window] > [File Name] from the menu bar causes the selected file to be displayed in the window on top. o Multiple files can also be displayed simultaneously on the same screen in three different ways: side by side horizontally, above and below vertically, and in a cascading fashion where the windows for each file are overlapping, but the edge of each is visible and may be selected to bring that file to the front. From the menu bar, select [Window] > [Tile Horizontal], [Window] > [Tile Vertical], or [Window] > [Cascade] as appropriate. [Executing Operations in an Existing File] o Data analysis, display, output, and other operations can be performed from the Result/Analysis screen, and settings and sample information can be changed from the Plate Setup screen. o Sample information and settings for an existing file may be changed and data analysis and other operations can be performed while a run is in progress for a new experiment file in the background. However, operations on the main unit, such as turning the halogen lamp OFF, cannot be performed. 23

26 o The PCR reaction conditions can be verified from the Thermal Profile Setup screen after a run is complete, but they cannot be changed. Also, a new run cannot be performed for the same file. Every time a run is performed, a new experiment file is created. [Saving Files] o To overwrite a file, select [File] > [Save] from the menu bar. o To save a file with a different file name, select [File] > [Save as] from the menu bar. The Save window is displayed. Select the destination folder, assign a file name, and save. o A confirmation message is displayed if a file with the same name already exists. [Closing Files] o Select [File] > [Close] from the menu bar, or click the button in the upper right corner. o If the experiment file has not been saved, a confirmation message is displayed asking whether or not to save the file. o The confirmation message is also displayed if changes have been made to an existing experiment file. Selecting Yes will save by overwriting the old file. Selecting No will close the file without saving any changes. If Cancel is selected, the file will not close. (3) Experiment File Management Using the Data Manager, it is possible to search for saved experiment files, verify file information, and change the destination folder for saving. [Experiment File Search and File Information Verification] a. Select Data Manager from the File menu. The Data Manager window is displayed. b. Select the folder to be searched from the tree view. c. Verify the folder in the search path and click the Search button. Check the search subfolder box if subfolders are also to be searched. d. The search results are displayed in the file list. e. Click the experiment type, user ID, or file name columns to sort the file lists according to one type of information. Clicking again causes the sorting order to switch from ascending to descending or vice versa. Click the Renew button to restore the original order. f. If a refined search is necessary, select the items to be refined from the experiment type or user ID combo box. 2. Software Overview 24

27 g. Selecting a file from the file list displays the information for the file at the bottom. File Path Displays the experiment file path From File Path Displays the folder path before the file is moved Experiment Type Displays Experiment Type User ID Displays User ID Create Time Displays the date and time the file was created Update Time Displays the date and time the file was last updated Description Displays memos entered in Description h. Click the Open button to open the selected experiment file. [Note] When the Data Manager window is opened with a new experiment file, the Load as Template buttons is displayed. Clicking the Load as Template button causes the Plate Setup and Thermal Profile Setup information for the selected file to be read into the new experiment file. [Changing the Save Destination Folder for Saving] a. Select an experiment file from the file list and drag it to the desired folder in the tree view. b. After the file is moved, the current path is displayed in file path and the former path is displayed in from file path. c. Click the Renew button to update the display. 25

28 3. Performing a Run 3-1. Experiment File Settings (1) Creating a New Experiment File Create a new experiment file to perform a run. a. Select New from the File menu. b. The New Experiment Options window is displayed. c. Select an Experiment Type. Absolute Quantification: Performs absolute quantification using a standard sample for which the absolute quantity is known. Relative Quantification: Performs a comparison of expression levels between samples. This method is mainly used for a gene expression analysis. Plus/Minus Assay: Plus/minus judgments are done for the target and the internal control respectively. Those results are combined to make an integrated judgment of Plus/Minus for the unknown sample. SNP Genotyping Assay: Plus/minus judgments are done for the allele1 type and the allele2 type respectively. Those results are combined to make an integrated judgment of Genotype for the unknown sample. Single PCR: Select single PCR when detecting one type of dye in one well. Single PCR should be selected even when detecting two or more types of dye in one plate if only one type of dye is detected in each well. Multiplex PCR: Select multiplex PCR when detecting two or more types of dye in one well. d. Select a user in the User ID field. [Note] If the user is not registered, register a new user in the Management Operator window (Refer to 2-4). e. Click the OK button when the settings are complete. (2) Changing the Experiment Type Normally the experiment type is set when a new experiment file is created, but the settings can be changed afterwards, if necessary. They can be changed at any time except when a run is being performed, but some of the settings must be changed in the Plate Setup screen. a. Display the Experiment Options screen. b. Click the Edit button to allow changes to the experiment type. c. Set the experiment type and data polarity. 3. Performing a Run 26

29 [Note] Selecting "-" from the Data Polarity menu causes sign inversion of the fluorescence value. This setting is used for the detection method in which the fluorescence value decreases as the amount of the PCR substance increases. d. Click the OK button after the settings are complete Set up the PCR Master Mix (1) Preparation of PCR Master Mix [96-Well Plate] a. Dispense the PCR master mix into each well. b. Hold both sides of the plate seal tab and peel off the white paper from the back. c. Place the seal on the plate. d. With the plate sealing pad, carefully smooth the seal to make it stick evenly to the plate. [Caution] If the seal does not adequately adhere to the plate, the solution may evaporate while a run is in progress, and measurements may not be performed accurately. e. Cut the tabs off both sides of the plate seal. f. Briefly centrifuge the plate to position the solution at the bottom of the well and remove air bubbles. Appropriate Inappropriate Reaction solution positioned at the bottom of well Reaction solution on side wall of well Air bubble at bottom of well 27

30 [8 Strip Tube] a. Dispense the PCR master mix into each tube. b. Close the tube cap securely. c. Briefly centrifuge the plate to position the solution at the bottom of the well and remove air bubbles. [Caution] Always use a flat-type cap. Never use a dome-type cap. [Caution] Do not touch the plate or 8 Strip tube with bare hands. This may cause fluorescence noise. (2) Setting up the Plate/Tube a. Verify that the STANDBY lamp is illuminated. b. Pull up the lid handle on the front of the main unit, and slide it towards the rear. [Caution] The handle is locked when a run is being performed. Do not try to force the lid open, because this may damage the equipment. If the cover needs to be opened while a run is being performed, click the Pause button. When the run is paused, the lock is released. c. Set the tube or plate containing the reaction solution onto the sample block. If using a plate, set it on the sample block so that the A1 well is in the far left corner. [Warning] The temperature of the lid rises to 100ºC or more during operation, and there is a danger of bodily injury from burns if it comes into contact with skin. Also, after the run is completed, the sample block may remain for some time at the final set temperature. In either case, take care not to touch hot parts of the equipment. d. Pull the handle forward, push it down, and close securely Setting the PCR Conditions PCR conditions and fluorescent data measurement conditions are set in the Thermal Profile Setup screen. Because it is not possible to change the settings after a run has started, make sure that the settings are correct before performing the run. (1) Basic Settings PCR conditions consist of patterns and segments. All patterns consist of single or multiple segments. Set the number of cycles per pattern. And set the temperature, the hold time, and whether or not to perform measurements on each segment. a. Clicking the Thermal Profile Setup button displays the Thermal Profile Setup screen. b. Set the filter to be used for measurement in the Collect Data field. All filters are selected by default (FAM and ROX are standard). [Note] Fluorescent filter data that is not checked in Data Collect is not saved. Therefore, it is not possible to change the fluorescent filter settings and perform the analysis again after the run is complete. Please select the filter that is appropriate for the experiment. It is especially essential in the case of multiplex PCR to select the correct filter. 3. Performing a Run 28

31 b c a c. In the Speed field, set the temperature control speed for the thermal cycler. The Fast setting is normally used by default. If amplification does not proceed well with the Fast setting, however, retry using the Normal setting. d. The setting conditions for each pattern and segment are displayed, showing visual charts and numerical values. Set the temperature and time for each segment after adding or deleting patterns as needed. e. Before adding a pattern, click the box in the uppermost section of the segment to assign an additional position. Select the pattern to be added from the Pattern menu in the lower part of the screen and click the Add Pattern button. Hold: Maintains a constant temperature. 2 Step PCR: Performs 2-Step PCR (shuttle PCR). 3 Step PCR: Performs 3-Step PCR. Dissociation: Performs dissociation curve analysis. Custom: Used when creating an original pattern. f. To delete a pattern, select the entire pattern to be deleted and click the Delete button. When the confirmation message is displayed, click the Yes button. g. If it is necessary to change the number of cycles, the temperature, or the Hold Time, double-click the corresponding numerical value in order to change the settings. [Note] A fixed time period for measurements is required in the segments in which the data is collected. A hold time of at least 12 seconds is necessary when the measurement is performed with two types of filter, and at least 8 seconds, with one type of filter. If the input time is less than the minimum hold times given above, a warning message is displayed, and the input time must be corrected. 29

32 h. Check the Data Collection box for the segments in which fluorescence measurement is to be performed. Remove the check if fluorescence measurement is not to be performed in a segment. [Note] For 2-step PCR, fluorescence measurement is conducted in the 2nd segment, while for 3-step PCR, fluorescence measurement is conducted in the 2nd or 3rd segment. If fluorescence measurement is to be conducted in two or more segments, create a pattern consists of Custom segments. With a custom pattern, you can check the Data Collection box of any segments. (Refer to Section (2) Advanced Settings.) i. If Dissociation is set as a pattern, the Dissociation Time input field at the top of the screen becomes active, and it is possible to change the time settings. [Note] For a dissociation curve analysis, the temperature is raised at 0.5ºC intervals and fluorescence measurement is performed at each temperature. The Dissociation Time is the hold time at each temperature. The default is set at the shortest time of 4 seconds. (2) Advanced Settings [Create Original Pattern] a. Select the position where the pattern is to be added. b. Select Custom from the Pattern menu, click the Add Pattern button, and the pattern is added. c. If the new pattern consists of multiple segments, continue adding segments until complete. d. Set the number of cycles in the Cycle field. e. Set the Temperature, Hold Time, and Data Collection for each segment. [Temperature and Time Increased/Decreased for Each Cycle] This is used when touchdown PCR is performed. a. When Extend is checked at the top of the screen, the extend function is displayed. b. Time Extension: If the hold time is to be extended for each cycle, input the extension time into the Increment Time field. 3. Performing a Run 30

33 c. Temperature Change: If the temperature is to be changed for each cycle, input the temperature increment/decrement in degrees in the Increment Temp field. [Note] The range of possible settings for Increment Time is 0 to 300 seconds. The range of possible settings for Increment Temp differs depending upon the temperature settings in the segment and the number of cycles. In either case, however, it is not possible to input a numerical value that is outside the range of possible settings. [Load Settings from an Existing Experiment File] The thermal profile of an existing file may be used as a template. a. Click the Load Template button to open the Open window. b. Select an experiment file and click Open. c. By performing the operations described above, edit the thermal profile as needed Run (1) Starting a run a. Set the reaction plate or tube onto the sample block of the main unit. b. Verify that the STANDBY indicator of the main unit is illuminated, and click the Start Run button at the lower right of the Thermal Profile Setup screen. c. If the experiment file has not been saved, the Save As window is displayed. Assign a save destination folder and file name and click the Save button. d. The buzzer of the main unit sounds once, and the run starts. While the run is being performed, the lid handle is locked and the lid cannot be opened. [Note] At first, pre-incubation is performed at 25ºC for one minute in order to stabilize the plate temperature. After that, the run starts according to the settings. [If the Run Does Not Start Immediately] If the main unit is still warming up or the lid is not completely closed, a warning message is displayed, and the run does not start. [Lid Temperature is Less Than 105ºC] a. A warning message is displayed when the Start Run button is clicked. b. Click OK and wait for the lid temperature to rise to 105ºC. This should take about five minutes. c. Verify that the STANDBY indicator is illuminated. Then click the Start Run button again to start the run. 31

34 [Halogen Lamp is Still Warming Up or OFF] a. When the Start Run button is clicked, a confirmation message is displayed. b. If the run is to be started without waiting for the lamp to warm up, click the Run Now button. [Note] If the Run Now button is clicked when the halogen lamp is OFF, the lamp lights up, and the run starts at once. c. If the run is to be performed after the lamp has finished warming up, click the Cancel button. When the lamp has warmed up, the STANDBY lamp is illuminated and the run may be started by clicking the Start Run button. [Note] If the Cancel is clicked while the halogen lamp is OFF, the lamp lights up and starts warming up. [Lid is not Closed] a. A warning message is displayed when the Start Run button is clicked. b. Click OK and close the lid completely. c. Click the Start Run button again to start the run. [If Normal PCR is to be performed with the Halogen Lamp OFF] When the machine is to be used as a normal thermal cycler for PCR, and there is no need for fluorescence measurement, a run can be performed with the halogen lamp OFF. a. In the Thermal Profile Setup screen, remove all checks for the Collect Data filters, or remove all the Data Collection checks in the thermal profile. b. Click the Start Run button. A confirmation message is displayed. c. If the run is to be performed without any data collection, click the Yes button to confirm. [Caution] If performing a run with the halogen lamp turned OFF, the Turn Lamp OFF after Run box in the side area is automatically checked. After the run is complete, the lamp remains OFF. (2) Monitoring Run Status The progress of the run may be verified. a. Time/Temperature, Running Point, and Instrument Control are displayed as additional buttons in the side area while the run is being performed. 3. Performing a Run 32

35 b. When the Time/Temperature tab is clicked, remaining time, lid temperature, and block temperature are displayed. Remaining Time: Time remaining until the run ends. Lid Temperature: The temperature of the lid. Block Temperature: The temperature of the sample block. c. When the Running Point tab is clicked, the numbers of the current pattern, segment and cycle are displayed. d. In the profile view, the current segment is displayed in orange. (3) Instrument Control during a Run While a run is being performed, it is possible to add cycles, pause and restart, forcibly terminate, and perform other operations. When the Instrument Control tab is clicked, the buttons for various controls are displayed. [Adding Cycles] a. Clicking the Add Cycle button displays the Add Cycle window. b. Input the number of cycles to be added in the Additional Cycle field. c. Click the OK button. [Caution] Addition of cycles is only possible during PCR execution. However, a cycle cannot be added during the last PCR cycle. If the Add Cycle button is clicked during the last PCR cycle or while on hold, a warning message is displayed. [Pause and Restart] a. When the Pause button is clicked, a confirmation message appears. b. Click the Yes button to pause the run at the current point. The buzzer of the main unit sounds three times, and the lid handle lock is released. At the same time, the Restart button becomes active. [Warning] During a pause, the lid and sample block remain at the temperature set before the Pause button was clicked. The lid and sample block may still be hot, and there is a danger of bodily injury from burns if they come in contact with skin. Take care not to touch either the lid or the sample block. c. Click the Restart button to resume the run. The buzzer of the main unit sounds once, the lid handle is locked, and the run resumes. [Skipping to Next Pattern] This operation terminates the pattern in progress and skips to the next pattern. When sufficient PCR amplification is verified, you can terminate the PCR and skip to the next dissociation curve analysis, if desired. 33

36 a. Click the Skip to Next button. A confirmation message is displayed. b. Click the Yes button to skip to the next pattern after the current cycle ends. [Forced Termination] This operation forces the run to terminate. Data from the run up to the point of the forced termination is saved and can be analyzed. However, after a forced termination operation, it is not possible to resume the run. a. Click the Stop button. A confirmation message is displayed. b. If the Yes button is clicked, the run is completed after the current cycle ends and a completion message is displayed. At this time the buzzer of the main unit sounds 6 times, and the lock of the lid handle is released. [Warning] Immediately after a forced termination, the lid and sample block of the main unit may be hot, and there is a danger of bodily injury from burns if these parts come in contact with skin. Be careful not to touch either the lid or the sample block. [Note] Clicking the close button in the top right corner of the experiment file does not terminate a run in progress. Instead, the following message is displayed. [Automatically Turning Lamp OFF After Run Completion] When the Turn lamp OFF after run box in the side area is checked, the lamp of the main unit turns off after the run is complete. If subsequent PCR operations are not required, it is recommended that this function be used in order to conserve the life of the halogen lamp. (4) Real Time Results Display a. Click the Result/Analysis button in the side area. The Result/Analysis screen is displayed. b. Click the Filter button for the results to be displayed (multiple filters can be selected). 3. Performing a Run 34

37 c. When the Full button is clicked, the selected data is displayed in a full screen. (Click the Dual button to return to the dual window display.) d. In the Selector, select the well for which the results are to be displayed. The Selector is displayed when the Selector tab is clicked. If the tab is clicked again, it is hidden. [Note] Refer to chapters 4 to 8 for details about the Result/Analysis screen, including the types of graph and the use of the Selector. (5) Run Completion The run ends automatically when all the thermal profile setting has been performed completely. a. When the thermal profile is completed, the buzzer of the main unit sounds six times, the lid lock is released, and the run ends. b. A run completion message appears. Verify and click the OK button. The run status display in the side area disappears. c. Open the lid handle and remove the reaction plate. [Warning] After run completion, the lid and sample block of the main unit may remain hot for some time, and there is a possibility of bodily injury from burns if the equipment comes in contact with skin. Therefore, take care not to touch the lid or sample block. d. Input the sample information in the Plate Setup screen, and perform analysis in the Results/Analysis screen. (6) Run History Verification From the run history a number of actions may be verified such as run start and end dates/times, pauses and restarts, forced terminations, and other information. a. Select [Analysis] > [Data Profile] from the menu bar to display the Data Profile window and verify the run history. b. Click the OK button to end. 35

38 4. Basic Operations 4-1. Plate Document Settings Enter the sample information in the Plate Setup screen. The settings may be set or changed while a run is being performed or after the run has ended. (1) Basic Operations a. Click the Plate Setup button in the side area. The Plate Setup screen is displayed. b. Making of lists Enter or select information in the Target List and Sample List. The asterisk (*) means those are essential items (Type*, Name*, etc.). Click Add button to increase Targets or Samples, and click Delete button to decrease them. [Note] Set the Target/Sample Type at one time If multiple targets or samples have the same Type, you can set it at one time. First, select all targets or samples with the same Type, and then select the Type from drop-down menu. c. After completing the lists, click Update button. This number of Targets or Samples will be added by clicking the Add button. d. Making of Plate Image Set the Target and Sample for each well on the Plate Image. (1) Select the corresponding wells. (2) Select the Target or Sample for the wells on the Plate Image Editor. 4. Basic Operations 36

39 (1) Select wells. (2) Select Target/Sample. After you finish the setting, click Hide Editor button to close the Plate Image Editor. At that time, Hide Editor button will change to Show Editor button. Click the button again to re-open the Plate Image Editor. [Note] Copy, Paste and Delete sample information You can copy and paste the sample information. And you can also undo or redo the operation. To delete the sample information, select the corresponding wells and press the delete key on the keyboard. Copy: Ctrl+C Undo: Ctrl+Z Paste: Ctrl+V Redo: Ctrl+Y (2) Convenient functions [Load Target/Sample List] You can load Target List and Sample List that you made outside this software. Those lists should be TSV (Tab Separated Values) text file. a. Making of Target List and Sample List You can set the following items in each list. <Target List> <Sample List> Annotation file Target List Description Format type Type* Target Type TOI or REF dye Dye* Dye FAM, ROX, etc. description Name* Target Name forward Forward Forward Primer sequence 3'tag-sequence-5'tag reverse Reverse Reverse Primer sequence 3'tag-sequence-5'tag probe Probe Probe sequence 3'tag-sequence-5'tag amplicon Amplicon amplicon sequence 3'tag-sequence-5'tag commercialassay Assay ID commercial assay ID "company:order number" xref xref reference ID "name of the database(reference ID)" Annotation file Sample List Description Format type Type* Sample Type UNKN, STD, NAC, NTC description Name/Std. Qty.* Sample Name/Standard quantity calibrator Calibrator Calibrator or not yes or no interruncalibrator interruncalibrator Inter run calibrator or not false or true dnasetreatment dnasetreatment DNase treated or not yes or no enzyme enzyme RT enzyme primingmethod primingmethod RT priming method oligo-dt, random, target specific thermconditions thermal cycling conditions ID rnaquantity rnaquantity RNA quantity "value unit" dnaquantity dnaquantity DNA quantity "value unit" rnaqualitymethod rnaqualitymethod RNA quality method dnaqualitymethod dnaqualitymethod DNA quality method rnaqualtity rnaquality RNA quality "value unit" dnaqualtity dnaquality DNA quality "value unit" 37

40 Make a Target/Sample List in TSV (Tab Separated Values) text file format. The first line of the list should be the items of Annotation file in the above table. And each of contents should be entered in the format specified above. It s convenient to use a Target/Sample annotation file as a template. To do so, export it from Output Setup screen (Refer to 9-3). <Example of making a list using text editor> <Example of making a list using Excel> * Save the file as TSV (Tab Separated Values) text file if you use Excel to make a list. [Note] If you use primers of Perfect Real Time Support System, you can export a Target List from the system. Refer to the help of Perfect Real Time Support System for the details. b. Click the Load button to open the File Open screen. c. Select the file you made in the step a. and click the Open button. d. If needed, edit the sample information. [Load Settings from Existing Experiment Files] Load the Plate settings of an existing experiment file as a Template. a. Click the Load Template button. The File Open window appears. b. Select an experiment file and click the Open button. c. If needed, edit the sample information. [Caution] Because input items differ according to the experiment type, it is not possible to load settings from a different file type. The following message is displayed if a different file type is selected. 4. Basic Operations 38

41 [ID/Name Display] Although sample information is displayed with names in default, you can also choose the display with IDs instead of names. a. Select the Name radio button to display with names. b. Select the ID radio button to display with IDs. [Auto Target/Sample] You can use Auto Target/Sample function to do the setting continuously. a. Set the first Target or Sample as usual. b. Click the Auto Target or Auto Sample button to activate the Auto function. c. During the Auto function is ON, the next ID is displayed at mouse pointer. d. Select the corresponding wells to set the next Target/Sample automatically. e. To stop the Auto function, click the Auto Target or Auto Sample button again. c. The next ID at mouse pointer. This ID will be set when you select wells. 39

42 4-2. Basic Operations of Analysis Graphs and the results of the analysis are shown on the Result/Analysis Screen. a. Click a Filter button to display the graph for that filter. FAM and ROX are the standard filters. It is also possible to select multiple filters in the same graph. a b c d b. Clicking the Full and Dual buttons switches the screen display back and forth from full screen to dual window display mode. c. Select the graphs to be displayed from the Analysis Data and Fluorescence menus. Amplification Plot: A primary curve with fluorescence values on the vertical axis and the number of cycles on the horizontal axis. Standard Curve: A standard curve with Ct values on the vertical axis and the initial template quantity of the standard sample on the horizontal axis. Relative Quantity Chart: Relative Quantity is displayed in a bar chart. Dissociation Curve: A dissociation curve with fluorescence values on the vertical axis and temperature on the horizontal axis. Scatter Plot: The distribution of data is displayed as a scatter plot. Plate Format: This figure displays the judgment result in a plate format. Text Report: Displays measurement data such as Ct and Tm values and sample information in a table format. d. Under the Selector tab, select the well to be displayed and analyzed. Clicking the Selector tab to show or hide it. e. In dual window display mode, checking the box at the top right of the Selector tab links the selected wells in the two windows. Each one can be selected independently if the check is removed. 4. Basic Operations 40

43 f. Well Selector or Target/Sample Type Selector may be chosen. Well Selector: Select the corresponding well(s) to display and analyze. Target/Sample Type Selector: Select all the wells of the same target or sample type by checking the box.. [Note] Using the right-click menu in Well Selector, you can change the kind of sample information displayed and also the omit settings. Select Target from View, show target number on the wells Changing the Graph Properties It is possible to change the settings for lines, symbols and X and Y scale in the graph. [Graph Operation from the Chart Properties Window] a. Select the graph type from the Analysis Data menu. b. Double-click the graph, or select Properties from the right-click menu on the graph. The Chart Properties window is displayed. c. The window contains the following three setting tabs: Axis & Plot Area: Settings for X-axis and Y-axis ranges, scale, and background color. Line: Settings for displaying/hiding lines and for line patterns, and thickness. Symbol: Settings for displaying/hiding symbols and for symbol shape, and size. Error Bar: Settings for displaying/hiding the error bars, and the pattern, and thickness of error bars for each replicate. Color: Verify color setting of each Target and Sample. Kinds of tab on the Chart Properties. Amplification Plots Axis&Plot Area [1] Line [4] Symbol [5] Color Standard Curve Axis&Plot Area [2] Symbol Line(STD) [6] Symbol(STD) [7] Color [8] Relative Quantity Chart Axis&Plot Area [3] Error Bar [9] Color Dissociation Curve Axis&Plot Area [1] Line [4] Symbol [5] Color * [ ] : Figuer number 41

44 [X axis, Y-axis and Background Color Settings] a. Click the Axis & the Plot Area tab in the Chart Properties window. b. To change the upper or lower limits of the X-axis range, remove the Auto Limit check and input a value in the input field. c. To change the upper or lower limits of the Y-axis range, remove the Auto Limit check and input a value in the input field. There are also two types of automatic settings for the axis ranges: Semi-Auto (default) and Full-Auto. Semi-Auto: Sets minimum/maximum values for all wells. Full-Auto: Sets minimum/maximum values for the wells selected in the Selector area. d. Select the Scale. Linear: Linear display (default). Log: Logarithmic display. e. Click the Edit button under Back Color to change the background color. The Color Setting window is displayed. Select a color and click the OK button. [1] [2] [3] f. At Label Rotation, set the axis label display angle (Standard Curve, Relative Quantity Chart only). g. At Label Display, select the axis label display contents (Relative Quantity Chart only). ID: To display ID Name: To display name h. At Label Lines, select the axis label display method (Relative Quantity Chart only). Single: Display as one line Multi: Display as two lines i. Click the OK button in the Chart Properties window when settings are complete. [Line Pattern, Thickness Settings] a. Click the Line tab in the Chart Properties window. b. Select Show or Hide. When Show is set, the graph will display the line. If Hide is selected, the line is not displayed, and the following changes cannot be carried out. c. The wells selected in the Selector area are displayed in the list. Clicking the Show All button displays all the wells. [4] d. The types of line patterns that can be set are shown below. To change the pattern, click on the Pattern column of the well that is to be changed in the list and select from the menu. Solid: Solid line (default). Dash: Dashed line. Dot: Dotted line. Dash Dot: Line of alternating dashes and dots. 4. Basic Operations 42

45 Dash Dot Dot: Line of dashes followed alternately by two dots. e. To change the line thickness, click on Thickness column of the well that is to be changed and select the line thickness from 1 to 10 (default is 2). f. Click the OK button in the Chart Properties window when the settings are complete. [Symbol Format, Size Settings] a. Click the Symbol tab in the Chart Properties window. b. Select Show to set a symbol to appear in the graph. When Hide is selected, a symbol is not displayed, and the following changes cannot be performed. c. The well selected in the Selector area is displayed. When the Show All button is clicked, all wells are displayed. d. The symbol shape can be selected from the 15 types in the chart below. To change the symbol, click on Shape column of the well in the list and select the desired shape from the menu. Box Star Circle Dot VerticalLine Square Tri HorizontalLine OpenTri Diamond Cross + OpenDiamond InvertedTri DiagCross OpenInvertedTri [5] e. To change the symbol size, click on Size column of the well that is to be changed and select a number (one to 16) from the menu. f. Click the OK button in the Chart Properties window when settings are complete. [Note] In the Chart Properties screen of Standard Curve, there are Line (STD) tab and Symbol (STD) tab besides the above-mentioned. In these tabs, you can set the properties of standard curve lines and symbols. [6] [7] 43

46 [Verification of Color Settings] a. Click the Color tab in the Chart Properties window. b. The wells selected using the Selector are displayed in the list. Click Show All button to display all the wells. c. Verify the settings and click the OK button. [8] [Verification of Error Bar Settings] a. Click the Error Bar tab in the Chart Properties window. b. Check Show to display the error bar in the graph. Check Hide to hide the error bar in the graph. c. The replicates selected using the Selector are displayed in the list. Clicking the Show All button displays all replicates. d. Verify the settings and click the OK button in the Chart Properties window. [9] [Scale Adjustment from the Axis Screen] Double-click on the X- or Y-axis on the graph to set minimum/maximum values for that axis. a. X-axis Maximum Double-click on the area that is approximately the highest 1/3 of the X-axis. The AxisX Max window is displayed. Uncheck the Auto box and input the new maximum value in the number field. Click the OK button to change the X-axis maximum to the number input. b. X-axis Minimum Double-click on the area that is approximately the lowest 1/3 of the X-axis. The AxisX Min box is displayed. Uncheck the Auto box and input the new minimum value in the number field. Click the OK button to change the X-axis minimum to the number input. c. Y-Axis Maximum and Minimum These operations are carried out in the same way as for the X-axis. 4. Basic Operations 44

47 [Graph Operation using the Right-Click Menu] Right-clicking on the graph displays shortcut menus for setting of some chart properties and opening the Chart Properties window. The setting items that may be selected via the right-click menu are shown in the following table. X Axis Max Auto Maximum value automatic settings Manual Maximum value manual settings Min Auto Minimum value automatic settings Manual Minimum value manual settings Scale Linear Linear display Log Logarithmic display Y Axis Max Auto Maximum value automatic settings Manual Maximum value manual settings Min Auto Minimum value automatic settings Manual Minimum value manual settings Scale Linear Linear display Log Logarithmic display Symbol Show Display Symbol Hide Hide Symbol Line Show Display Line Hide Hide Line Error Bar Show Display Error Bar Hide Hide Error Bar Properties Display Chart Properties window 45

48 5. Absolute Quantification o o Absolute Quantification is a method to analyze the absolute quantity of an unknown sample based on a standard curve created by using a standard sample for which the absolute quantity is known. There are four different sample types that can be set in an Absolute Quantification experiment: UNKN (Unknown), STD (Standard), NTC (No Template Control) and NAC (No Amplification Control) Plate Document Settings [Absolute Quantification (Single)] a. Making of Target List Enter or select information in the Target List. The asterisk (*) means those are essential items. Make the list as described bellow or load it from a file you made beforehand outside this software (Refer to 4-1(2)). (1) Click Add button to increase Targets, and click Delete button to decrease them. (2) If needed, change the Dye. Select from the pull down menu. (3) Enter the Target Name. (4) If needed, change the Color. b. Making of Sample List Enter or select information in the Sample List. The asterisk (*) means those are essential items. Make the list as described bellow or load it from a file you made beforehand outside this software (Refer to 4-1(2)). (1) Select the Type from the pull down menu. NTC (No Template Control): Control reaction performed without template. NAC (No Amplification Control): Control reaction performed without enzyme (RTase or DNA polymerase). STD (Standard): Sample of known concentration to make a standard curve. UNKN (Unknown): Sample of interest whose quantity will be determined. 5. Absolute Quantification 46

49 (2) Enter the sample name or standard initial quantity in the Name/Std. Qty. (2) For Standards, enter initial quantity. The number will be displayed as exponents. (3) If needed, change the Color. c. After completing the Target List and Sample List, click Update button. d. Making of Plate Image (1) Select the corresponding wells on the Plate Image and select the target on the Plate Image Editor. (2) Select the corresponding wells on the Plate Image and select the sample on the Plate Image Editor. First, select wells Next, select target/sample. 47

50 e. After completing the setting, the Plate Image should be like the figure below. [Note] Omit setting Select wells which should be omitted from the analysis and click the Omit button. Those wells will be marked with X like the figure below. Fluorescent data of all of the 96 wells is measured regardless of the omit setting while executing the run. Therefore, if you reset the omit setting, you can use those data for the analysis again. [Absolute Quantification (Multiplex)] The setting methods are similar to those of Absolute Quantification (Single). The differences of Single and Multiplex are mainly described here. a. Making of Target List (1) Click Add button to increase Targets, and click Delete button to decrease them. (2) Select the Dye from the pull down menu. (3) Enter the Target Name. (4) If needed, change the Color. 5. Absolute Quantification 48

51 b. Making of Sample List (1) Select the Type from the pull down menu. (2) Enter the sample name or standard initial quantity in the Name/Std. Qty. (3) If needed, change the Color. c. After completing the Target List and Sample List, click Update button. d. Making of Plate Image (1) Select the corresponding wells on the Plate Image and select the target on the Plate Image Editor. Change the filter by clicking the filter button and set the target for other dyes. Set the target of FAM on the FAM screen. Set the target of ROX on the ROX screen. (2) Select the corresponding wells on the Plate Image and select the sample on the Plate Image Editor. In case of Multiplex PCR, since the same well contain the same sample inevitably, the same sample will be automatically entered for all dyes. Automatically set the same sample 49

52 [Note] It is necessary to set the correct type of fluorescent dye in order to perform accurate crosstalk correction. If the fluorescent dye shown on the button is not the dye actually in use, change the Dye Name using the Filter Settings. (Refer to 2-4(3).) 5-2. Analysis (1) Analysis Parameter Settings When the run is complete, the optimal analysis parameters are set automatically. Verify the analysis results. If the analysis was not performed correctly using the auto settings, select the parameters manually and perform the analysis again. For the Crossing Point Method, set the baseline and threshold for the Primary Curve. For the 2nd Derivative Maximum Method, set the threshold for the 2nd Derivative. Crossing Point Method: A method designating the Ct value as the intersection point of the Primary Curve and the threshold. 2nd Derivative Maximum Method: A method designating the Ct value as the maximum point of the 2nd Derivative of the Primary Curve. [Primary Curve Baseline Settings] a. Select Amplification Plot from the Analysis Data menu. b. Select Primary Curve from the Fluorescence menu to display the Analysis Setting. c. In the Analysis Setting area, select the Filter to be used for the analysis. d. Click the Baseline tab to display the baseline setting items. Set the same cycle range for all wells: e. Select Manual and input the appropriate Start and End values. Click the Apply button to refresh the graph display. Set a different cycle range for each well: f. Click the Edit Individually button to display the Baseline Setting window. g. For each well to be set, select Manual from the drop-down menu of the A/M (Auto/Manual) column. An asterisk is displayed at the left of the row corresponding to the well that is being edited. h. Double-click the Start or End cell of the well that is to be set and input a numerical value. i. Click the Apply button. 5. Absolute Quantification 50

53 j. Click the OK button when the settings are complete. [Note] The baseline is set to the initial cycle range during no amplification is detected. In cases the Primary Curve startup position varies greatly among wells, apply an optimal range for each well rather than apply the same baseline range to all to give more accurate results. Please note that accurate baseline correction cannot be performed when settings are applied for a range that includes the startup portion. In the case of Auto settings, an optimum baseline range is set for each well. Example of a correct baseline setting Example of an insufficient baseline setting Example of an excessive baseline setting [Primary Curve Threshold Settings] There are two kinds of threshold for the Primary Curve, horizontal and vertical. Horizontal threshold will be used to calculate the Ct values. Vertical threshold is set to exclude the noisy region from the analysis, in case there are fluorescent fluctuations at the beginning of the cycles. If there are multiple targets, the threshold can be set to the optimum position of each target or a position common to all targets. Select either method by checking or clearing the check box To each target as follows: Individual setting by target Common setting to all targets a. Select Amplification Plot from the Analysis Data menu. b. Select Primary Curve from the Fluorescence menu. c. Change the Y-axis scale to log. Right-click on the graph and select [Y axis] > [Scale] > [Log]. Alternatively, double-click on the graph to display the Chart Properties window and set the Y-axis scale to Log. 51

54 d. In the Analysis Setting area, select a Filter for the analysis. e. Click the Threshold tab to display the threshold setting items. f. When Manual is selected, the input field on the right becomes active. g. Input a numerical value, or drag the threshold line on the graph up or down to set it. Click the Apply button to refresh the graph display. [Note] Set the horizontal threshold within the exponential phase of the Primary Curve (the range inside which the Primary Curve becomes a straight line when Y-axis displayed in Log scale). Example of an appropriate threshold Example of a threshold that is too low Example of a threshold that is too high [2nd Derivative Threshold Settings] Using the 2nd Derivative Maximum Method, the 2nd Derivative peak position is calculated as the Ct value. The thresholds for 2nd Derivative are set in order to avoid miscalculating the noise level peak as the Ct value. The method of setting the horizontal and vertical threshold is the same as Primary Curve. a. Select Amplification Plot from the Analysis Data menu. b. Select 2nd Derivative from the Fluorescence menu. c. In the Analysis Setting area, select the Filter for the analysis. d. Select Manual and input a numerical value in the input field, or click and drag the threshold line on the graph to move it. e. Click the Apply button to refresh the graph display. 5. Absolute Quantification 52

55 Example of an appropriate threshold Example of a threshold that is too low Example of a threshold that is too high [Smoothing Settings] A smoothing process is performed using a moving average method in order to decrease the fluctuation of measurement and the noise of each cycle. With default setting, the data of five points are averaged. If desired, this point-number can be changed to any odd-number value from one to nine. A moving average is not performed if the point number is one. a. Select [Analysis] > [Smoothing] from the menu bar. b. The Smoothing Setting window is displayed. c. From the drop-down menus beside Amplification Averaging Points and Dissociation Averaging Points, select the number of points to average. d. Click the OK button after the settings are complete. [Analysis Data Set Selection] This software displays and analyzes one set of PCR data (and one set of dissociation data) at once. Usually, analysis can be performed using the default settings, but when fluorescence is measured with multiple segments, or when dissociation measurement is repeated multiple times, it is necessary to assign the data set to analyze. a. With the default setting, the segment for which data collection was first performed is displayed and analyzed. b. Select [Analysis] > [Analysis Data Selection] from the menu bar to display the Analysis Data Selection window. The Pattern Number and Segment Number used for the analysis are displayed. c. Click Segment 3 in the Amplification area to change the data set to be analyzed from Segment 2 to Segment 3. The Pattern Number and Segment Number are changed. 53

56 d. Click the OK button to close the Analysis Data Selection window and change the graph display. [Caution] If a segment that has not collected the data is selected, the Pattern Numbers and Segment Numbers are not shown, and a graph is not displayed. (2) Analysis Results Display [Primary Curve and Ct Value] a. Select Amplification Plot from the Analysis Data menu. b. Select the curve for analysis from the Fluorescence menu. Raw: The measured fluorescence value (raw data) Primary Curve: The primary curve that results from the baseline and fluorescence value collection. 2nd Derivative: The 2nd Derivative function of the Primary Curve. c. Adjust the analysis parameter in the Analysis Setting area. (Refer to 5-2(1).) d. Verify the numeric data using the Text Report. (Refer to 5-2(3).) 5. Absolute Quantification 54

57 [Standard Curve and Quantity] a. Select Standard Curve from the Analysis Data menu. b. Select Crossing Point or 2nd Derivative Max from the Fluorescence menu. c. Verify the numeric data using the Text Report. (Refer to 5-2 (3).) Standard Curve Setting It is possible to use the standard curve of previous run for the analysis. a. Click the Set STD Curve button to show the Standard Curve Setting screen. b. Select Manual from D/M list and enter the Slope and Intercept of the standard curve. c. Click Apply button to reanalyze the data. [Note] Standard Curve Label o Displays the standard curve numerical formula, the coefficient of determination (R2), and the PCR efficiency (Eff). The basic arithmetic expression is Y = A Log (X) + B where Y = Ct value, X = Initial template quantity, and A and B are factors obtained in regression analysis. o For Multi-Target PCR, the standard curve is displayed for each target. Click the check box to switch display or hide for each of them. o The label can be moved to the optimal position in the graph by clicking the tab on the left. o Close the label by clicking the Hide button. To show the label again, select [Standard Curve Label] > [Filter Type (FAM or ROX)] > [Show] from the right-click menu. [Dissociation Curve and Tm value] a. Select Dissociation from the Analysis Data menu. b. Select the data to be displayed from the Fluorescence menu. Raw: The measured fluorescence values (raw data). 1st Derivative: The negative derivative of the measured fluorescence values. 55

58 c. Verify the numeric data using the Text Report. (Refer to 5-2(3).) (3) Text Report Text Report includes the sample information that was set in the Plate Document and the analysis data such as Ct and Tm value. a. Select Text Report in the Analysis Data menu. The Text Report is displayed in the graph display area. b. Select the well to be displayed in the Selector. c. Select the data set to be displayed from the Data Set menu. Data Set of Each Well: Displays the analysis results (Ct value, Tm value, etc.) for each individual well. Data Set of Replicate: Displays the analysis results (mean value, standard deviation, etc.) for each Replicate (a group in which the target and sample setting is the same). Select All: Displays all analysis results. d. Select the items to be displayed in the Show Items check box (multiple selections are possible). Analysis Setting: Items concerning analytical parameters. CP Method Data: Results obtained from analysis by the Crossing Point Method. SDM Method Data: Results obtained from analysis by the 2nd Derivative Maximum Method. e. Use the check box for each item in the Column list to display or hide that item in the Text Report. f. Click an item name in the Text Report to sort the data by that item. 5. Absolute Quantification 56

59 [Text Report Display Items] Well Sample ID Sample Name Sample Type Target ID Target Name Filter Fluorescence Last (Raw) Fluorescence Last Threshold (CP) Threshold (SDM) Baseline A/M Baseline Start Baseline End Ct (CP) Ct Avg. (CP) Ct SD (CP) Ct (SDM) Ct Avg. (SDM) Ct SD (SDM) Init Qty Qty (CP) Qty Avg. (CP) Qty SD (CP) Qty (SDM) Well position number Sample ID Sample name Sample Type (UNKN, STD, NTC) Target (gene) ID Target (gene) name Filter used for measurements Fluorescence raw data for the last cycle Primary Curve Fluorescence Values for the last cycle Threshold used for calculation of Ct value using CP Method Threshold used for calculation of Ct value using SDM Method Automatic (A) or manual (M) baseline settings Baseline range: First cycle Baseline range: Last cycle Ct values for individual wells calculated using CP method Average of Ct value of replicates calculated using CP method Standard deviation of Ct value of replicates calculated using CP method Ct values for individual wells calculated using SDM method Average of Ct value of replicates calculated using SDM method Standard deviation of Ct value of replicates calculated using SDM method Initial template quantity of the standard sample Quantity for individual wells calculated using CP Method Standard Curve Average of replicates of the quantity value calculated using CP Method Standard Curve Standard deviation of replicates of the quantity calculated using the CP Method Standard Curve Quantity for individual wells calculated using SDM Method Standard Curve Qty Avg. (SDM) Average of replicates of the quantity calculated using SDM Method Standard Curve Qty SD (SDM) Standard deviation of replicates of the quantity calculated using the SDM Method Standard Curve R2(CP) Coefficient of determination of CP Method Standard Curve Slope(CP) Slope of the CP Method Standard Curve (coefficient A) Efficiency(CP) R2(SDM) PCR efficiency calculated using CP Method Standard Curve Coefficient of determination of SDM Method Standard Curve Slope(SDM) Slope of the SDM Method Standard Curve (coefficient A) Efficiency(SDM) Tm #1 Tm #2 Tm #3 PCR efficiency calculated from the SDM Method Standard Curve Tm value calculated from the maximum peak of the Dissociation Curve Tm value calculated from the second highest peak of the Dissociation Curve Tm value calculated from the third highest peak of the Dissociation Curve 57

60 6. Relative Quantification o Relative Quantification is a method of analysis that determines the change in gene expression in a given sample relative to another reference sample, primarily for use in gene expression analysis. o Settings are performed for four sample types: STD (Standard), UNKN (Unknown), NTC (No Template Control) and NAC (No Amplification Control). In addition, a Reference is set to normalize the amount of RNA and a Calibrator is set as a control of unknowns. o Two different methods can be selected for performing the relative quantification analysis: the Standard Curve method and the ΔΔCt method Plate Document Settings In the Relative Quantification Assay, in addition to the items of the Absolute Quantification, a Reference is set to normalize the amount of RNA and a Calibrator is set as a control of unknowns. [Relative Quantification (Single)] a. Making of Target List Enter or select information in the Target List. The asterisk (*) means those are essential items. Make the list as described bellow or load it from a file you made beforehand outside this software (Refer to 4-1(2)). (1) Click Add button to increase Targets, and click Delete button to decrease them. (2) Select the Type from the pull down menu. REF (Reference): Reference gene to normalize the amount of RNA (housekeeping gene) TOI (Target Of Interest): Target gene to be quantified (3) If needed, change the Dye. Select from the pull down menu. (4) Enter the Target Name. (5) If needed, change the Color. b. Making of Sample List Enter or select information in the Sample List. The asterisk (*) means those are essential items. Make the list as described bellow or load it from a file you made beforehand outside this software (Refer to 4-1(2)). 6. Relative Quantification 58

61 (1) Select the Type from the pull down menu. NTC (No Template Control): Control reaction performed without template. NAC (No Amplification Control): Control reaction performed without enzyme (RTase or DNA polymerase). STD (Standard): Sample of known concentration to make a standard curve. UNKN (Unknown): Sample of interest whose quantity will be determined. (2) Enter the sample name or standard initial quantity in the Name/Std. Qty. (2) For Standards, enter initial quantity. The number will be displayed as exponents. (3) For Unknowns, select yes or no in the Calibrator setting. (3) For a control sample of unknowns, select yes in the Calibrator setting. (4) If needed, change the Color. c. After completing the Target List and Sample List, click Update button. d. Making of Plate Image (1) Select the corresponding wells on the Plate Image and select the target on the Plate Image Editor. (2) Select the corresponding wells on the Plate Image and select the sample on the Plate Image Editor. * The star marks ( ) are displayed in the wells of Reference (REF) and the circle marks ( ) in the wells of Calibrator. 59

62 First, select wells Next, select target/sample. e. After completing the setting, the Plate Image should be like the figure below. [Note] Omit setting Select wells which should be omitted from the analysis and click the Omit button. Those wells will be marked with X like the figure below. Fluorescent data of all of the 96 wells is measured regardless of the omit setting while executing the run. Therefore, if you reset the omit setting, you can use those data for the analysis again. 6. Relative Quantification 60

63 [Relative Quantification (Multiplex)] The setting methods are similar to those of Relative Quantification (Single). The differences of Single and Multiplex are mainly described here. a. Making of Target List (1) Click Add button to increase Targets, and click Delete button to decrease them. (2) Select the Type from the pull down menu. (3) Select the Dye from the pull down menu. (In case of Multiplex PCR, REF and TOI should be detected with different Dyes.) (4) Enter the Target Name. (5) If needed, change the Color. b. Making of Sample List (1) Select the Type from the pull down menu. (2) Enter the sample name or standard initial quantity in the Name/Std. Qty. (3) For Unknowns, select yes or no in the Calibrator setting. (4) If needed, change the Color. c. After completing the Target List and Sample List, click Update button. d. Making of Plate Image (1) Select the corresponding wells on the Plate Image and select the target on the Plate Image Editor. In case of Relative Quantification (Multiplex), since TOI and REF should be detected in the same well, REF will be set automatically in the same well when you set TOI. Automatically set REF. (2) Select the corresponding wells on the Plate Image and select the sample on the Plate Image Editor. In case of Multiplex PCR, since the same well contain the same sample inevitably, the same sample will be automatically entered for all dyes. 61

64 Automatically set the same sample [Note] It is necessary to set the correct type of fluorescent dye in order to perform accurate crosstalk correction. If the fluorescent dye shown on the button is not the dye actually in use, change the Dye Name using the Filter Settings. (Refer to 2-4(3).) 6-2. Analysis (1) Analysis Parameter Settings When the run is complete, the optimal analysis parameters are set automatically. Verify the analysis results. If the analysis was not performed correctly using the auto settings, select the parameters manually and perform the analysis again. The Primary Curve baseline and threshold settings, 2nd Derivative threshold settings, etc., operate in the same way as for Absolute Quantification. (Refer to 5-2(1).) (2) Analysis Results Display The Amplification Plot and Ct Value, Standard Curve and Quantity, and Dissociation Curve functions can all be operated using the same method as for Absolute Quantification. (Refer to 5-2(2).) [Relative Quantification] a. Select the Relative Quantity Chart from the Analysis Data list. b. The Relative Quantity is displayed as a bar graph with the error bar of standard deviation. The X-axis represents target ID sample ID. c. Select the Ct value calculation method from the RQ Based on drop-down menu. 6. Relative Quantification 62

65 d. Select the Relative Quantification analysis method in the Method box. Standard Curve Method: Analysis method using a standard curve. ΔΔCt Method: Analysis method that does not use a standard curve, but converts the Ct value difference to a relative quantification value. Standard Curve Setting It is possible to use the standard curve of previous run for the analysis. 1) Click the Set STD Curve button to show the Standard Curve Setting screen. 2) Select Manual from D/M list and enter the Slope and Intercept of the standard curve. 3) Click Apply button to reanalyze the data. [Note] This setting is the same with the one in the Standard Curve graph. And these settings will be reflected to each other. e. Verify the numeric data using the Text Report. (Refer to 6-2(3).) [Note] Target/Sample Label o The target and sample information are displayed on different labels. o By clicking the tab on the left of the label, it can be moved to any position on the graph. o Close the label by clicking the Hide button. To display it again, select [Target/Sample Label] > [Target] > [Show] or [Target/Sample Label] > [Sample] > [Show] from the right-click menu. (3)Text Report Text Report includes the sample information that was set in the Plate Document and the analysis data such as Ct and Tm value. The operation method is the same as for Absolute Quantification. (Refer to 5-2(3).) [Text Report Display Items] Well Sample ID Sample Name Sample Type Target ID Target Name Filter Fluorescence Last (Raw) Fluorescence Last Threshold (CP) Threshold (SDM) Baseline A/M Baseline Start Well position number Sample ID Sample name Sample type (UNKN, STD, NTC, NAC) Target (gene) ID Target (gene) name Filter used for measurement Fluorescence raw data for the last cycle Primary Curve Fluorescence Values for the last cycle Threshold used for calculation of Ct Value using CP Method Threshold used for calculation of Ct Value using SDM Method Automatic (A) or manual (M) baseline settings Baseline range: First cycle 63

66 Baseline End Ct (CP) Ct Avg. (CP) Ct SD (CP) Ct (SDM) Ct Avg. (SDM) Ct SD (SDM) Init Qty Qty (CP) Qty Avg. (CP) Qty SD (CP) Qty (SDM) Qty Avg. (SDM) Qty SD (SDM) Rel. Qty (CP) Rel. Qty SD (CP) Rel. Qty (SDM) Rel. Qty SD (SDM) ΔΔCt Rel. Qty (CP) ΔΔCt Rel. Qty SD+ (CP) ΔΔCt Rel. Qty SD-(CP) ΔΔCt Rel. Qty (SDM) ΔΔCt Rel. Qty SD+ (SDM) ΔΔCt Rel. Qty SD-(SDM) R2(CP) Slope(CP) Efficiency(CP) R2(SDM) Slope(SDM) Efficiency(SDM) Tm #1 Tm #2 Tm #3 Baseline range: Last cycle Ct value calculated for individual wells using the CP Method Average of Ct value of replicates obtained using the CP Method Standard deviation of Ct value of replicates obtained using the CP method Ct value calculated for individual wells using the SDM method Average of Ct value of replicates obtained using the SDM method Standard deviation of Ct value of replicates obtained using the SDM method Initial template quantity of standard sample Quantity for individual wells obtained using the standard curve of the CP method Average of quantity of replicates obtained using the standard curve of the CP method Standard deviation of quantity of replicates obtained using the standard curve of the CP method Quantity for individual wells obtained using the standard curve of the SDM method Average of the quantity of replicates obtained using the standard curve of the SDM method Standard deviation of the quantity of replicates obtained using the standard curve of the SDM method Average of relative quantity of replicates calculated from Ct values obtained using the CP method Standard deviation of relative quantity of replicates calculated from Ct values obtained using the CP method Average of relative quantity of replicates calculated from Ct values obtained using the SDM method Standard deviation of relative quantity of replicates calculated from Ct values obtained using the SDM method Average of relative quantity of replicates calculated using the ΔΔCt method on Ct value obtained using the CP method Upper limit value of relative quantity of replicates calculated using the ΔΔCt method on Ct value obtained from the CP method Lower limit value of relative quantity of replicates calculated using the ΔΔCt method on Ct value obtained from the CP method Average of relative quantity of replicates calculated using the ΔΔCt method on Ct value obtained from the SDM method Upper limit value of relative quantity of replicates calculated using the ΔΔCt method on Ct value obtained from the SDM method Lower limit value of relative quantity of replicates calculated using the ΔΔCt method on Ct value obtained from the SDM method Coefficient of determination of the standard curve for the CP method Slope (Coefficient A) of the standard curve for the CP method PCR efficiency obtained from the standard curve for the CP method Coefficient of determination of the standard curve for the SDM method Slope (Coefficient A) of the standard curve for the SDM method PCR efficiency obtained from the standard curve for the SDM method Tm value obtained from the highest peak of the dissociation curve Tm value obtained from the second highest peak of the dissociation curve Tm value obtained from the third highest peak of the dissociation curve 6. Relative Quantification 64

67 7.Plus/Minus Assay o o o Plus/Minus Assay is an analysis that judges whether a target gene exists in an unknown sample. There are three different sample types that can be set in the Plus/Minus Assay: UNKN (Unknown), PC (Positive Control), and NC (Negative Control). Plus/minus judgments are done for the target and the internal control respectively. Those results are combined to make an integrated judgment of plus/minus for the unknown sample Plate Document Settings In Plus/Minus Assay, the internal controls can be used to check the possibility of false negative(s). And the data of negative controls are used to set the threshold for the result judgment. [Plus/Minus (Single)] a. Making of Target List Enter or select information in the Target List. The asterisk (*) means those are essential items. Make the list as described bellow or load it from a file you made beforehand outside this software (Refer to 4-1(2)). (1) Click Add button to increase Targets, and click Delete button to decrease them. (2) If needed, change the Dye. Select from the pull down menu. (3) Enter the Target Name. (4) If needed, change the Color. b. Making of Sample List Enter or select information in the Sample List. The asterisk (*) means those are essential items. Make the list as described bellow or load it from a file you made beforehand outside this software (Refer to 4-1(2)). (1) Select the Type from the pull down menu. UNKN (Unknown): Samples to be analyzed. PC (Positive Control) NC (Negative Control) (2) Enter the Sample Name. (3) If needed, change the Color. 65

68 c. After completing the Target List and Sample List, click Update button. d. Making of Plate Image (1) Select the corresponding wells on the Plate Image and select the target on the Plate Image Editor. (2) Select the corresponding wells on the Plate Image and select the sample on the Plate Image Editor. e. After completing the setting, the Plate Image should be like the figure below. [Plus/Minus (Multiplex)] The setting methods are similar to those of Plus/Minus (Single). The differences of Single and Multiplex are mainly described here. a. Setting of Internal Control Dye Select the dye to detect the internal control from the pull down menu. b. Making of Target List (1) Click Add button to increase Targets, and click Delete button to decrease them. (2) Select the Dye from the pull down menu. 7. Plus/Minus Assay 66

69 (3) Enter the Target Name. (4) If needed, change the Color. c. Making of Sample List (1) Select the Type from the pull down menu. (2) Enter the Sample Name. (3) If needed, change the Color. d. After completing the Target List and Sample List, click Update button. e. Making of Plate Image (1) Select the corresponding wells on the Plate Image and select the target on the Plate Image Editor. (2) Select the corresponding wells on the Plate Image and select the sample on the Plate Image Editor. (3) Set the presence of Internal Control of each well. IC (-): Include the Internal Control IC (+): Not include Internal Control 67

70 7-2. Analysis (1) Analysis Parameter Settings When the run is complete, the optimal analysis parameters are set automatically. Verify the analysis results. If the analysis was not performed correctly using the auto settings, select the parameters manually and perform the analysis again. The Primary Curve baseline and threshold settings, 2nd Derivative threshold settings, etc., operate in the same way as for Absolute Quantification. (Refer to 5-2(1).) [Scatter Plot Threshold Settings] If there are multiple targets, the threshold can be set to the optimum position of each target or a position common to all targets. Select either method by checking or clearing the check box To each target. a. Select Scatter Plot from the Analysis Data menu. b. Select data from the Based on menu. c. In the Analysis Setting area, select the Filter to be used for the analysis. d. Click the Threshold tab to display the threshold setting items. e. When Manual is selected, the input field on the right becomes active. Input a numerical value, or drag the threshold line on the graph up or down to set it. f. Click the Apply button to refresh the graph display. [Advanced Setting] This setting can be used in an experiment that includes more than two negative controls and SD is not zero. a. Select Auto of Threshold settings and click the Advanced tab. b. Enter a numerical value from 1 to 99. c. Click the Apply button to refresh the graph display. 7. Plus/Minus Assay 68

71 (2) Analysis Results Display The Amplification Plot and Ct Value and Dissociation Curve functions can all be operated using the same method as for Absolute Quantification. (Refer to 5-2(2).) [Scatter Plot] a. Select Scatter Plot from the Analysis Data menu. b. Select data from the Based on menu. c. Verify the numeric data using the Text Report. (Refer to 7-2(3).) [Display Results in Plate Format] a. Select Plate Format from the Analysis Data menu. b. Select data from the Based on menu. The results are displayed in Plate Format by the following mark. +: Detected (the value is above the threshold) -: Not detected (the value is equal to or below the threshold) c. Check the box of Result to display the judgment. (This option is not displayed when the internal control filter is selected.) OK: Controls that reacted correctly OUT: Controls that reacted incorrectly Posi.: Positive Nega.: Negative ND: Not Detected (Both of the Target and Internal Control were not detected.) ERROR: Different judgments in the same replicate d. Verify the numeric data using the Text Report. (Refer to 7-2(3).) [Note] Display the well information In the Plate Format, only the results are shown in default. If you check the Well Information box, the well information also would be shown. And you can select each items is displayed or not with the checkbox of them. 69

72 (3)Text Report Text Report includes the sample information that was set in the Plate Document and the analysis data such as Ct and P/M result. The operation method is the same as for Absolute Quantification. (Refer to 5-2(3).) [Text Report Display Items] Well Sample ID Sample Name Sample Type Target ID Target Name Filter Fluorescence Last (Raw) Fluorescence Last Threshold (CP) Threshold (SDM) Baseline A/M Baseline Start Baseline End Ct (CP) Ct Avg. (CP) Ct SD (CP) Ct (SDM) Ct Avg. (SDM) Ct SD (SDM) Detected(F) Detected(CP) Detected(SDM) SP Threshold(F) SP Threshold(CP) SP Threshold(SDM) P/M Result (F) P/M Result (CP) P/M Result (SDM) Well position number Sample ID Sample name Sample Type (UNKN PC NC) Target (gene) ID Target (gene) name Filter used for measurement Fluorescence raw data for the last cycle Primary Curve Fluorescence Values for the last cycle Threshold used for calculation of Ct Value using CP Method Threshold used for calculation of Ct Value using SDM Method Automatic (A) or manual (M) baseline settings Baseline range: First cycle Baseline range: Last cycle Ct value calculated for individual wells using the CP Method Average of Ct value of replicates obtained using the CP Method Standard deviation of Ct value of replicates obtained using the CP method Ct value calculated for individual wells using the SDM method Average of Ct value of replicates obtained using the SDM method Standard deviation of Ct value of replicates obtained using the SDM method +/- for each filters based on Primary Curve Final +/- for each filters based on Primary Curve Ct +/- for each filters based on 2nd Derivative Ct Threshold used for Scatter Plot based on Primary Curve Final Threshold used for Scatter Plot based on Primary Curve Ct Threshold used for Scatter Plot based on 2nd Derivative Ct Plus/Minus judgment based on Primary Curve Final Plus/Minus judgment based on Primary Curve Ct Plus/Minus judgment based on 2nd Derivative Ct 7. Plus/Minus Assay 70

73 8.SNP Genotyping Assay o o o SNP Genotyping Assay is an analysis that judges a SNP genotype of an unknown sample. There are three different sample types that can be set in the SNP Genotype Assay: UNKN (Unknown), PC (Positive Control), and NC (Negative Control). Plus/minus judgments are done for the allele1 type and the allele2 type respectively. Those results are combined to make an integrated judgment of genotype for the unknown sample Plate Document Settings In the SNP Genotyping Assay, it is necessary to select the filter for Allele1 type and Allele2 type respectively. And the data of negative controls are used to set the threshold for the result judgment. a. Dye setting Select the dye for Allele1 type and Allele2 type respectively from the pull down menu. b. Making of Target List Enter or select information in the Target List. The asterisk (*) means those are essential items. Make the list as described bellow or load it from a file you made beforehand outside this software (Refer to 4-1(2)). (1) Click Add button to increase Targets, and click Delete button to decrease them. (2) Enter the Target Name. (3) If needed, change the Color. b. Making of Sample List Enter or select information in the Sample List. The asterisk (*) means those are essential items. Make the list as described bellow or load it from a file you made beforehand outside this software (Refer to 4-1(2)). (1) Select the Type from the pull down menu. UNKN (Unknown): Samples to be analyzed. PC(Allele1): Positive Control of Allele1 type PC(Allele2): Positive Control of Allele2 type NC (Negative Control) (2) Enter the Sample Name. (3) If needed, change the Color. 71

74 c. After completing the Target List and Sample List, click Update button. d. Making of Plate Image (1) Select the corresponding wells on the Plate Image and select the target on the Plate Image Editor. (2) Select the corresponding wells on the Plate Image and select the sample on the Plate Image Editor Analysis (1) Analysis Parameter Settings When the run is complete, the optimal analysis parameters are set automatically. Verify the analysis results. If the analysis was not performed correctly using the auto settings, select the parameters manually and perform the analysis again. The Primary Curve baseline and threshold settings, 2nd Derivative threshold settings, etc., operate in the same way as for Absolute Quantification. (Refer to 5-2(1).) To set the Scatter Plot threshold, operate in the same way as for Plus/Minus Assay. (Refer to 7-2(1).) 8. SNP Genotyping Assay 72

75 (2) Analysis Results Display The Amplification Plot and Ct Value and Dissociation Curve functions can all be operated using the same method as for Absolute Quantification. (Refer to 5-2(2).) [Scatter Plot] a. Select Scatter Plot from the Analysis Data menu. b. Select data from the Based on menu. c. Verify the numeric data using the Text Report. (Refer to 8-2(3).) [Display Results in Plate Format] a. Select Plate Format from the Analysis Data menu. b. Select data from the Based on menu. The results are displayed in Plate Format by the following mark. +: Detected (the value is above the threshold) -: Not detected (the value is equal to or below the threshold) c. Click the Genotype button to display the judgment results. OK: Controls that reacted correctly OUT: Controls that reacted incorrectly Allele1: Allele1 type Allele2: Allele2 type Hetero: Hetero type ND: Not Detected (Both of the Allele1 type and Allele2 type were not detected.) ERROR: Different judgments in the same replicate [Note] Display the well information In the Plate Format, only the results are shown in default. If you check the Well Information box, the well information also would be shown. And you can select each items is displayed or not with the checkbox of them. 73

76 (3)Text Report Text Report includes the sample information that was set in the Plate Document and the analysis data such as Ct and P/M result. The operation method is the same as for Absolute Quantification. (Refer to 5-2(3).) [Text Report Display Items] Well Sample ID Sample Name Sample Type Target ID Target Name Filter Fluorescence Last (Raw) Fluorescence Last Threshold (CP) Threshold (SDM) Baseline A/M Baseline Start Baseline End Ct (CP) Ct Avg. (CP) Ct SD (CP) Ct (SDM) Ct Avg. (SDM) Ct SD (SDM) Detected(F) Detected(CP) Detected(SDM) SP Threshold(F) SP Threshold(CP) SP Threshold(SDM) SNP Genotype(F) SNP Genotype (CP) SNP Genotype (SDM) Well position number Sample ID Sample name Sample Type (UNKN PC(Allele1) PC(Allele2) NC) Target (gene) ID Target (gene) name Filter used for measurement Fluorescence raw data for the last cycle Primary Curve Fluorescence Values for the last cycle Threshold used for calculation of Ct Value using CP Method Threshold used for calculation of Ct Value using SDM Method Automatic (A) or manual (M) baseline settings Baseline range: First cycle Baseline range: Last cycle Ct value calculated for individual wells using the CP Method Average of Ct value of replicates obtained using the CP Method Standard deviation of Ct value of replicates obtained using the CP method Ct value calculated for individual wells using the SDM method Average of Ct value of replicates obtained using the SDM method Standard deviation of Ct value of replicates obtained using the SDM method +/- for each filters based on Primary Curve Final +/- for each filters based on Primary Curve Ct +/- for each filters based on 2nd Derivative Ct Threshold used for Scatter Plot based on Primary Curve Final Threshold used for Scatter Plot based on Primary Curve Ct Threshold used for Scatter Plot based on 2nd Derivative Ct SNP Genotype judged based on Primary Curve Final SNP Genotype judged based on Primary Curve Ct SNP Genotype judged based on 2nd Derivative Ct 8. SNP Genotyping Assay 74

77 9. Exporting and Printing 9-1. Exporting The Plate Document, Thermal Profile, Result/Analysis Graphs and Text Reports, as well as numerical value data, can be output in various file formats. (1) Plate Document and Thermal Profile a. Display the Plate Setup Screen or the Thermal Profile Setup Screen. b. Right-click in either the Plate Document or Thermal Profile to display the menu below. Select Plate Setup and Right-click Select Thermal Profile Setup and Right-click [Bitmap Image Output] c. Select Output Image. d. The Save As screen is displayed. e. Select the save destination folder, assign a file name, and click Save. [MS Word and PowerPoint Format File Output] Basic information such as the file name is output together with MS Word or PowerPoint files and can be used for data management. c. Select either Word or PowerPoint from the Make Report menu. d. The Save As screen is displayed. e. Select the save destination folder, assign a file name, and click Save. [PDF Format File Output] c. Select Print. d. The Report Print Screen is displayed. Verify the contents using various tools. e. Select Save from the Report Print screen menu bar or toolbar. f. The Save As screen is displayed. g. Select the save destination folder, assign a file name, and click Save. 75

78 (2) Graphs Every graph displayed on the Result/Analysis Screen can be output as an image file. a. Select Analysis Data and Fluorescence to display a graph. b. Right-clicking on the graph displays the menu shown at right. [Copy to Clipboard] c. Select Copy. The figure now displayed will be copied to the clipboard. d. You can paste the figure to any other application software. [Bitmap File Output] c. Select [Output Image] > [Just This State]. d. The Save As screen is displayed. e. Select the save destination folder, assign a file name, and click Save. [Bitmap File Output (when setting size)] c. Select [Output Image] > [Specify Image Size]. d. Set the width, height, and font size in the Specify Image Size screen. e. Click the OK button to apply the settings. f. The Save As screen is displayed. g. Select the save destination folder, assign a file name, and click Save. [Note] Clicking Change Default assigns the current setting as the default value for all subsequent operations. This function may be used to ensure that subsequent images are saved with the same image size. [MS Word and PowerPoint Format File Output] Basic information such as the file name is output together with MS Word or PowerPoint files and can be used for data management. c. Select either Word or PowerPoint from the Make Report menu. d. The Save As screen is displayed. e. Select the save destination folder, assign a file name, and click Save. [PDF Format File Output] c. Select Print. d. The Report Print Screen is displayed. Verify the contents using various tools. e. Select Save from the Report Print screen menu bar or toolbar. f. The Save As screen is displayed. g. Select the save destination folder, assign a file name, and click Save. 9. Exporting and Printing 76

79 (3) Numerical Data Numerical data based on the graphs displayed in the Result/Analysis screen can also be output. In addition, the analysis results are consolidated into a Text Report in tabular format. [Output of Numerical Value Data from a Graph] a. Select Analysis Data and Fluorescence to display the graph. b. Right-click on the graph to display the menu. c. Select [Export Data] > [CSV] or [Excel]. d. The Save As screen is displayed. e. Select the save destination folder, assign a file name, and click Save. [Text Report Output] a. Select Text Report from Analysis Data. b. Right-click on the Text Report to display the menu. c. Select [Export Data] > [CSV] or [Excel]. d. The Save As screen is displayed. e. Select the save destination folder, assign a file name, and click Save. [Copy the Text Report] a. Select Text Report from Analysis Data. b. Right-click on the Text Report to display the menu. c. Select cells of Text Report you want to copy. d. Select Copy. The data of selected cells will be copied to the clipboard. e. You can paste the data to other application software such as Excel. (4) Reports Reports that consolidate information from several graphs, including the Plate Document, Thermal Profile, and Analysis Results, can be created. a. Select [File] > [Make Full Report] to display the Report Setting window. b. Check the boxes for the Plate Setup and/or Thermal Profile Setup that are to be included in the report. c. For Result/Analysis, check the boxes for graphs to be included in the report (Amplification Plots, Standard Curve, Relative Quantity Chart, and/or Dissociation Curve). d. Under Fluorescence, select either Primary Curve or 2nd Derivative. 77

80 e. If the Relative Quantity Chart is checked, check the StdCrv and/or ΔΔCt. f. Select either Word or PowerPoint as the file format. g. Verify the contents and click the OK button. h. Select the save destination folder in the Save As Screen, assign a file name, and click Save. [Note] To create a report, it is necessary to select beforehand a filter for analysis in the Result/Analysis screen. If a filter is not selected, a warning message is displayed Printing The Plate Document, Thermal Profile, and Result/Analysis Graphs may be printed as a Report. (1) Plate Document and Thermal Profile a. Display the Plate Setup Screen or the Thermal Profile Setup Screen. b. Right-click in either the Plate Document or Thermal Profile to display the menu below. c. Select Print. d. The Report Print screen is displayed, and the contents can be verified using various tools. e. Select Print from the menu bar or toolbar in the Report Print screen. f. The Print screen for the printer driver that is in use is displayed. Carry out the necessary settings, and click OK. g. After printing is complete, click the close button at the top right of the Report Print screen to close it. (2) Graphs Graphs of the analysis results can be printed. a. Select Analysis Data and Fluorescence to display a graph. b. Right-click on the graph to display the menu. c. Select Print. d. The Report Print screen is displayed, and the contents can be verified using various tools. e. Select Print from the menu bar or toolbar in the Report Print screen. f. The Print screen for the current printer driver is displayed. Carry out the necessary settings, and click OK. g. Close the Report Print screen after printing is complete. 9. Exporting and Printing 78

81 (3) Reports Reports that consolidate information from several graphs, including the Plate Document, Thermal Profile, and Analysis Results, can be created and printed. a. Select [File] > [Make Full Report] to display the Report Setting window. b. Select graphs to be included in a report. (Refer to 9-1(4)) c. Verify the contents and click OK button to make a report. d. The Report Print screen is displayed, and the contents can be verified using various tools. e. Select Print from the menu bar or toolbar in the Report Print screen. f. The Print screen for the current printer driver is displayed. Carry out the necessary settings, and click OK. g. Close the Report Print screen after printing is complete. [Note] The report can also be saved as a PDF format file. Select Save As from the menu bar or toolbar in the Report Print screen. The Save As screen is displayed. Select a save destination folder, assign a file name, and click Save Export files for RDML Select files you need for making of RDML file, and export them. a. If needed, change the Run ID. b. Select Detection Method, CP or SDM. c. Select files you want to export. d. If needed, select items to be exported. e. Click Output button to export the files. d. Select items by each check box. c. Select files by each check box. 79

82 10. Hardware Settings Halogen Lamp The halogen lamp on the main unit can be turned ON and OFF using the software. The software also allows the user to verify when the halogen lamp was last replaced and the cumulative time of usage. As a rough standard, the lamp should be stable and usable for about 1000 hours. After 1000 hours have been exceeded, a message is displayed recommending that the lamp be replaced. (1) Turning the Lamp ON/OFF o To turn the halogen lamp ON, select [Instrument] > [Lamp On] from the menu bar. To turn the lamp OFF, select [Instrument] > [Lamp Off]. The lamp can also be switched ON and OFF by clicking the icon in the toolbar. o When turning the lamp OFF, a confirmation message appears. (2) Lamp Status Verification and Reset after Replacement a. Open the Lamp Status screen by selecting [Instrument] > [Lamp Status] from the menu bar. b. Verify the lamp status. Total Time of Usage: Displays cumulative time of use. Date Last Replaced: Date when the lamp was last replaced. c. If the lamp has been replaced, click the Reset button, and Total Time of Usage is reset. Click the Yes button when the confirmation message is displayed. d. Verify that the total time and date information has changed, and click the Close button. 10.Halogen Lamp 80