iQ™5 Optical System Software Instruction Manual Compatible with the MyiQ™2, MyiQ™, and iQ™5 real-time PCR detection systems
NOTICE TO PURCHASER Bio-Rad’s real-time thermal cyclers are licensed real-time thermal cyclers under Applera’s United States Patent 6,814,934 B1 for use in research, human in vitro diagnostics, and for all other fields except the field of veterinary diagnostics. Purchase of this instrument conveys a limited nontransferable immunity from suit for the purchaser’s own internal research and development and for use in applied fields under one or more of U.S.
Safety Information Grounding Always connect the MyiQTM2, MyiQTM, or iQTM5 optics module to a three-prong, grounded AC outlet using the AC power cord provided with the system. Do not use an adaptor to a two-terminal outlet. Always ensure that you set the module power switch to the off position when you connect or disconnect power cords. Handling Handle all components of the real-time PCR detection system with care and with clean, dry hands at all times.
Table of Contents NOTICE TO PURCHASER ..................................................................................................i Safety Information .........................................................................................................ii Section 1. Getting Started ..............................................................................................1 1.1 The MyiQ™2 Real-Time PCR Detection System ................................................................1 1.
4.1.4 Selected Data File Area ..........................................................................................21 4.2 Plate Setup ..................................................................................................................21 4.2.1 Plate Setup Editor Window .....................................................................................21 4.2.2 Well Definition Icons ..............................................................................................22 4.2.
5.5 Show Plate Window .....................................................................................................44 5.6 Monitor Run Window....................................................................................................45 5.7 Run-Time Protocol Editing ............................................................................................45 Section 6. Data Analysis Module ..................................................................................47 6.1 PCR Quant Tab .
6.7.7 Vertical Threshold ..................................................................................................74 6.7.8 Horizontal Threshold ..............................................................................................74 6.7.9 Normalize Data ......................................................................................................74 6.7.10 Restore Default....................................................................................................75 6.
7.2 Components Required for Calibration .......................................................................... 111 7.3 Preparing Calibration Plates ........................................................................................ 112 7.4 Performing Calibrations .............................................................................................. 112 7.4.1 Performing Mask Alignment .................................................................................. 113 7.4.
Appendix A: Warranty ................................................................................................136 Appendix B: Troubleshooting Error Messages ...........................................................137 Appendix C: Product Ordering Information ...............................................................
Section 1 Getting Started Section 1. Getting Started This section contains information on the following topics: o The MyiQ2 Real-Time Detection System (page 1) o Setting up the system hardware (page 1) o Installing the iQ5 software (page 4) o Recommended computer settings (page 4) o Compatibility with earlier versions of the software (page 6) 1.
Section 1 Getting Started iQ5 Optical Accessory Kit (catalog #170-9752) • Optical reaction block • Modified sliding rear cover for iCycler thermal cycler • Serial cable • USB cable • Filter extraction tool • Optical tape applicator tools (3) • Optics support bracket • Support bracket screws • Hex driver • Hex screws • Spare part: halogen lamp MyiQ2 Calibrator Dye Solution Kit (catalog #170-8791) • 1x calibration dye solutions (4 dyes, 3 tubes of each dye) External Well Factor Soluti
Section 1 Getting Started 1.2.3 Installing the Support Bracket A support bracket with roller is provided for the MyiQ2, MyiQ, or iQ5 optics module. It is mounted to the rear of the iCycler thermal cycler. Align the optics module support bracket with the two holes on the rear of the iCycler thermal cycler. Using a #2 Phillips screwdriver, adjust the height of the bracket with two of the appropriate screws provided with the system accessories.
Section 1 Getting Started module is installed on the iCycler chassis. This connection senses when the lid handle is lifted. 1.3 Installing the iQ5 Software Locate the software installation disk for iQ5 Optical System software version 2.1 provided with the MyiQ2 system. This installation disk is compatible with computers running the Windows XP and Windows Vista 32-bit operating system. 1. Insert the iQ5 Optical System software installation CD in a CD-ROM drive. 2.
Section 1 Getting Started Computer Power Management Settings on Windows Vista 1. From the Start menu, choose Control Panel. Switch the Control Panel view to Classic View. 2. Choose Power options, and then choose Create a Power Plan. 3. Choose the High Performance plan, then enter the Plan name iQ5 software, and press Next. 4. Change the Turn Off The Display and Put the Computer to Sleep options to “Never”. 5. Click Save Changes. 6.
Section 1 Getting Started 1.5 Calibrating the Instrument Before using the real-time PCR detection system for the first time, mask alignment, background calibration, persistent well factor collection, and pure dye calibration (for MyiQ2 and iQ5 systems only) must be performed. See section 7, Calibrating the Instrument, for detailed instructions on calibration. 1.
Section 2 Quick Guides Section 2.
Section 2 Quick Guides Editing or Creating a Protocol Edit the selected protocol by clicking Edit in the Selected Protocol window, or create a protocol from a protocol template by clicking Create New in the Selected Protocol window. NOTE: Clicking Edit or Create New in the Selected Protocol pane opens the Protocol Editor. NOTE: You can only exit the Protocol Editor by clicking Save & Exit Protocol Editing or Cancel & Exit Protocol Editing. 1.
Section 2 Quick Guides window. Click Edit to open the plate setup in the Plate Setup Editor or • Click Data File and select the desired data setup file from the file tree directory. Click the file name to open the plate setup associated with the data file in the bottom right section of the Workshop window. Click Edit to open the plate setup in the Plate Setup Editor 2. Enter or edit any notes about the plate setup in the Notes box. 3. Enter or edit the sample volume, seal type, and vessel type. 4.
Section 2 Quick Guides 2. From the Workshop Setup window, select your desired Plate (.pts) and Protocol (.tmo) file individually, or your desired Run Set (.run). 3. Click Run. 4. Check that the desired Protocol and Plate Setup are displayed in the bottom half of the Initiate Run screen. 5. Select the type of well factors to use by selecting either: • Use Persistent Well Factors or • Collect Well Factors from Experimental Plate 6. Click Begin Run. 7.
Section 2 Quick Guides 2. Select a data file from the file tree browser, and then click Analyze. The file opens in the PCR Quant tab within the Data Analysis module. 3. Select or deselect wells to be included in the analysis by selecting Analyze Wells. Select or deselect wells to be displayed by selecting Display Wells. 4. The iQ5 software automatically chooses the data analysis conditions including baselines and thresholds.
Section 2 Quick Guides 3. Select the wells to analyze by clicking Analyze Wells. 4. Define the positive and/or negative controls in the Define Controls column within the End Point Analysis table. 5. Click Recalculate. The End Point Analysis table displays a positive, negative, or blank label for each unknown under the Unknowns Call column. 6. Click Reports to obtain customized reports of the End Point Analysis. End Point Analysis of an Existing Data File 1.
Section 2 Quick Guides 7. Choose between the Threshold Cycle and RFU display modes in the Display Mode area to display the allelic discrimination data. • Threshold Cycle displays the distribution of samples on the scatter plot based on the threshold cycle (CT). Samples that do not cross threshold will be assigned the CT value of the last cycle run in the experiment. • RFU displays the distribution of samples on the scatter plot based on the RFU generated by each sample at the last PCR cycle number.
Section 2 Quick Guides • Change the gene assignments in these wells by typing your desired name into the gene pull-down men, then click enter to apply the name to the selected wells • Change the condition assignments in these wells by typing your desired name into the condition pull-down menu, then click enter to apply the name to the selected wells NOTE: The Gene and Condition Names have a character entry limit of 15 characters • Minimize the Gene Expression Plate interface by clicking on the “-” but
Section 2 Quick Guides • Minimize the Gene Expression Plate interface by clicking on the “–” button to return to the standard view of the Gene Expr tab window 4. Click the Settings tab, then select Gene List to set your desired reference gene(s). 5. Click Normalized Expression (ddCt). 6. Click Recalculate to see your results. 7. Normalized expression results are graphed. The Data Table spreadsheet is accessed by clicking Data Table.
Section 2 Quick Guides • Change the condition assignments in these wells by typing your desired name into the condition pull-down menu, then click enter to apply the name to the selected wells NOTE: The Gene and Condition Names have a character entry limit of 15 characters. • Minimize the Gene Expression Plate interface by clicking on the “–” button to return to the standard view of the Gene Expr tab window 7. Select either Normalized expression (ddCt) or Relative quantity (dCt).
Section 2 Quick Guides 8. Click a sample type icon. 9. Select the type of replicate loading desired. 10. Click or drag across the plate to define wells with the selected fluorophore and sample type. 11. Continue defining the remaining wells that will contain the first fluorophore by changing to other sample type icons as appropriate.
Section 2 Introduction to the iQ5 Optical System Software Section 3. Introduction to the iQ5 Optical System Software (Version 2.1) The iQ5 software is divided into five sections, called modules. Icons representing each of the modules are always shown on the left side of the screen. The active or selected module has an orange background, whereas unselected modules have a gray background. Each module is subdivided into windows that perform a specific function for that module.
Section 4 Workshop Module Section 4. Workshop Module This section contains information on the following topics: o Setup Tab (page 19) o Plate Setup (page 21) o Protocol (page 31) o Run Set (pg 37) o Opening a Data File (page 39) o Applying Alternate Calibration Files (page 39) The Workshop module consists of the Setup and Plate Summary tabs. For detailed information on the information contained in the Plate Summary tab, refer to section 4.2.10 4.1 Setup Tab The Setup tab window (Figure 4.
Section 4 Workshop Module 4.1.1 File Browser Section Located in the top left of the Setup window, the file browser area contains a folder tree on the left side and a file list on the right side. The files displayed in the list depend upon the selected Setup window tab. With the Setup tab active, the displayed Setup window contains four additional tabs: • Protocol: Used to select, edit, or create a protocol for running a real-time experiment.
Section 4 Workshop Module Fig. 4.2. The Selected Plate Setup Area. 4.1.4 Selected Data File Area Located in the top right of the Setup window, the Selected Data File area displays the selected data file or the run set file name that you selected in the file browser area. The Notes box displays the notes associated with either the run set or data file. Buttons in this area include: • Run: Used to initiate a real-time PCR experimental run. • Begin End Point: Used to initiate an End Point run.
Section 4 Workshop Module Fig. 4.3. The Plate Setup Editor Window. Exiting the Plate Setup Editor You can return to the Workshop at any time by pressing Cancel & Exit Plate Editing. Any current changes made since the file was last saved will be lost. To save changes before returning to the home screen, click Save & Exit Plate Editing and assign a name to your new plate setup in a standard Windows Save dialog box. All plate setup files are automatically assigned an extension of .pts.
Section 4 Workshop Module Defines Positive Controls Paint Bucket icon: When the Paint Bucket icon is active and you click a well in the plate setup, all wells in that replicate group will be assigned the currently selected fluorophore. Delete Fluorophore icon: When the Delete Fluorophore icon is active and you click in a well in the plate setup, the currently selected fluorophore is removed from all wells in that replicate group. It does not change the definition of the sample type in that well.
Section 4 Workshop Module 9. Click a fluorophore. 10. Click or drag across the plate to define wells with the selected fluorophore and sample type. 11. Continue defining the remaining wells that will contain the first fluorophore by changing to any other sample type icons required. To calculate standard concentrations automatically, click Dilution Series and enter the upper or lower concentrations and units of the standards, set the dilution factor, and click Apply Dilution Series. 12.
Section 4 Workshop Module Fig. 4.6. List of Five Sets of Wells. Click Select/Add Fluorophores to open the Fluor Selector box. From this box, choose up to five fluorophores to be monitored. To remove a fluorophore from the list to be monitored, uncheck the box next to its name. To change the default color associated with a fluorophore, click the color box on the row with the fluorophore name and choose a new color as shown below in Figure 4.7. Fig. 4.7.
Section 4 Workshop Module 4.2.5 Whole Plate Loading There are two pieces of information required for each well: sample type and fluorophore(s) to be monitored. There are two ways of providing that information to the plate setup editor. When Whole Plate Loading is selected, you specify the sample type and fluorophores separately.
Section 4 Workshop Module The Horizontal and Vertical Replication buttons, together with the number in the Size box, specify the direction and number of replicates to be automatically defined. With the horizontal direction (row) button active and the Size set at 5, if you: • Click a well, the sample type is incremented by one for the next five wells in the horizontal direction, and each of the five wells will contain an identical replicate number.
Section 4 Workshop Module Fig. 4.8. The Seal and Vessel Type Dialog Box. 4.2.8 Defining a Dilution Series The iQ5 software can automatically calculate the concentrations of each member of a dilution series, given the dilution factor and the concentration of either the most or least concentrated sample (Figure 4.9). Fig. 4.9. The Dilution Series Dialog Box. 1. Access the dilution series box by clicking on Dilution Series. 2. Define the range of standard replicates to be defined.
Section 4 Workshop Module To assign values to any newly added standards while Define is not selected, you must open the Dilution Series dialog box again, make any necessary edits, and click Apply Dilution Series. 4.2.9 Plate Setup Editor Spreadsheets There are two different spreadsheets available from the Plate Setup Editor window: the Well Identifier and the Plate Setup Editor Spreadsheets. Well Identifier Spreadsheet The Well Identifier spreadsheet (Figure 4.
Section 4 Workshop Module replicate group. Replicate group assignment changes should be made on the plate, not in the spreadsheets. This single-dye layer spreadsheet has another feature that differentiates it from the single-well spreadsheet. From this spreadsheet you may Import Well Identifiers from an external comma separated values (CSV) file. Importing Well Identifiers From the Plate Editor spreadsheet you may import well/sample identifiers from an external CSV file.
Section 4 Workshop Module 4.3 Protocol Protocol files direct the operation of the iCycler thermal cycler and also specify when optical data will be collected during the thermal cycling run. iQ5 software protocol files are stored with the extension .tmo.
Section 4 Workshop Module Fig. 4.13. The Protocol Selected Protocol Pane. 4.3.2 Editing or Creating a Protocol To edit a selected protocol, double-click the protocol file or click Edit in the Selected Protocol window. To create a protocol from a protocol template, click Create in the Selected Protocol window. Clicking Edit or Create in the Viewing Protocol window will transfer the software to the Protocol Editor.
Section 4 Workshop Module The Protocol Editor may only be exited by clicking Save & Exit Protocol Editing or Cancel & Exit Protocol Editing. 4.3.3 Add Protocol Options If you want to add any protocol options, first enable them by clicking in the check box next to its description in the Show Options box. The Protocol Options include: • Gradient (see section 4.3.
Section 4 Workshop Module Temperature Change You may program an automatic periodic increase or decrease in the step temperature in a repeated cycle. Temperature increments or decrements may be as little as 0.1°C per cycle. You may make the increase or decrease as frequently as every cycle, and the increase or decrease can begin following any cycle.
Section 4 Workshop Module 4.3.4 Gradient A thermal gradient may be programmed across the reaction block at any step of a protocol. The gradient runs from the back of the instrument to the front, with the hottest temperature in row A and the coolest temperature in row H. All wells in each respective row are at the same temperature so at any time during a gradient step, there will be eight different temperatures across the block with 12 wells at each temperature.
Section 4 Workshop Module Programming a Specific Temperature for a Specific Row To program a specific temperature in any single row: 1. Click Gradient in the Show Options box. Two columns will appear in the spreadsheet and a representation of the gradient will appear on the right side of the window. 2. Click the Gradient checkbox in the spreadsheet for the desired step. 3. Enter the desired temperature into a specific single row on the gradient display. 4. Press Enter. 5.
Section 4 Workshop Module Fig. 4.16. Melt Curve Protocol Editing Table. 6. Anneal curves can be also created by entering the temperature change as a negative value (for example, –0.5). 7. Save the protocol by entering a file name and then clicking Save & Exit Protocol Editing. NOTE: The minimum dwell times necessary for data collection are 10 seconds for 1 fluorophore. We recommend using a slightly higher dwell time than the minimum values so that more data points are collected at each repeat. 4.3.
Section 4 Workshop Module Fig. 4.17. The Run Set Selection Window. 4.4.1 Selecting a Run Set 1. Click Run Set. 2. Use the browser below the Run Set button to locate the folder containing the run set file , and then click on the file name to select it. 3. The selected run set will appear in the Selected Protocol and Selected Plate Setup windows. 4.4.2 Creating a Run Set To create a run set: 1. Select the plate setup and protocol that are to be linked. 2.
Section 4 Workshop Module 4.5.1 Selecting a Data File 1. Click the Data File button. 2. Use the file browser below the Data File button to locate the folder that contains the data file, and then click on the file name to select it. 3. The protocol and plate setup used in the data file will appear in the Selected Protocol and Selected Plate Setup windows. The plate setup that is shown can be either the Original or Current (last saved) plate setup.
Section 4 Workshop Module 4. After the RME calibration file has been opened a Save Optical Data File dialog box will appear. The existing pure dye calibration will be overwritten. NOTE: It is recommended to save the optical data file with a different name as this will ensure that the original file, with the original calibration data, is always maintained for reference. 4.5.
Section 5 Run-Time Central Module Section 5. Run-Time Central Module This section contains information of the following topics: o Initiating a Run (page 41) o Well Factors (page 42) o Real-Time PCR experiments using binding dyes (page 43) o Running End Point experiments (page 43) o Monitoring a Run (page 45) o Run-Time Protocol Editing (page 45) The Run-Time Central module is entered automatically after clicking Run or Run End Point from the Workshop Setup window (Figure 5.1).
Section 5 Run-Time Central Module Fig. 5.2. The Initiate Run Window. 5.2 Well Factors Well Factors are used to compensate for any system or pipeting non-uniformity in order to optimize fluorescent data quality and analysis. Well Factors may be collected directly from the experimental plate (dynamic well factors) or from an external well factor plate (persistent well factors).
Section 5 Run-Time Central Module Persistent well factors are stored in the Well Factors folder in the iQ5 Folder. Refer to section 7 for information on the calibration and generation of persistent well factors. In general, persistent well factors can be used for approximately three months, but should be regenerated anytime something pertinent to the optical system is changed such as the optical filters or the camera lamp.
Section 5 Run-Time Central Module Fig. 5.3. The End Point Setpoint Box. To begin a run, click Begin Run. The Save dialog box opens. Type a name for the optical data file. The iQ5 software saves data automatically during the experiment. 5.5 Show Plate Window The Show Plate window (Figure 5.4) can be used to visualize samples loaded into the reaction block of the system.
Section 5 Run-Time Central Module 3. If a multiplex assay is being performed, repeat steps 1 & 2 for all fluors present in Fluor Selection list. 4. When finished, return to the Initiate Run screen. 5.6 Monitor Run Window Open the Monitor Run window by clicking the Monitor Run tab. The window appears as shown in Figure 5.5. Traces are displayed in real-time and run progress is monitored in the top left hand section of the window. Fig. 5.5. The Monitor Run Window. 5.
Section 5 Run-Time Central Module • • Add 10 Repeats: Click Add 10 Repeats to add additional repeats to the current cycle. You can click this button multiple times, however, the total number of repeats is limited to 600. For example, it may be necessary to add repeats to a run in an experiment amplifying low copies of DNA to allow all samples to cross threshold. Click Add 10 Repeats to an amplification cycle of 30 repeats to make it 40 repeats.
Section 6 Data Analysis Module Section 6.
Section 6 Data Analysis Module • Gene Expr: The Gene Expression screen has flexible tools for the determination of the fold induction of one gene relative to another gene or relative to itself under different circumstances, for example, temporally, geographically, or developmentally different points • Edit Plate: In the Edit Plate screen, you may make changes to the sample type assignment or to the quantities of the Standards in the plate setup used to run the experiment.
Section 6 Data Analysis Module The PCR Quant tab consists of three sections: • Amplification chart • Standard Curve chart • Results section 6.1.1 Customizing the PCR Quant Display You can customize the size of the sections in the PCR Quant tab in a number of ways. In the upper corner of each section is a + (plus) button that enlarges the section when clicked. The enlarged section has a – (minus) button that reduces the section when clicked.
Section 6 Data Analysis Module Use the fluorophore selector buttons to select and deselect which fluorophores to display. Deselecting a fluorophore removes it from the display, but not from the analysis, selected fluorophore buttons are displayed with a red border around their perimeter. De-selected fluorophores are displayed without this red border. In Figure 6.3, the FAM fluorophore has been selected, and HEX has been de-selected. Fig. 6.3. Fluorophore Selector Buttons. 6.2.
Section 6 Data Analysis Module 6.2.4 Selecting and Viewing Traces • Identifying a specific amplification trace: Identify a specific trace by moving the mouse pointer along the trace until the hand icon appears. The dialog box identifies the trace, by both well name and fluorophore, in the top left corner of the amplification chart • Selecting a specific amplification trace: Select a specific trace by moving the mouse pointer on the trace until the hand icon appears, and then double-click.
Section 6 Data Analysis Module Removing wells from data analysis changes the calculation of the threshold location. Changing the calculation of the threshold location may result in a change in the threshold crossing (CT) location, thus affecting all subsequent data analyses that depend on CT values. This would include standard curve calculations and quantification of unknowns, gene expression, and allelic discrimination using threshold crossing values.
Section 6 Data Analysis Module Fig. 6.6. The Select Analyzed Wells for Display Window. To select the wells to include in data display: 1. In the PCR Quant tab, click Display Wells. The Select Analyzed Wells for Display window appears. Only those wells that are included in the analysis appear in the window. 2. Select and de-select wells using the same methods described in the analyze wells section on page 51. 3.
Section 6 Data Analysis Module • The slope of the line • The y-intercept You can enlarge the standard curve chart by clicking on the plus (+) sign in the upper left corner of the section. Fig. 6.7. The Standard Curve Chart. 6.3.1 Standard Curve Chart Menu Right-clicking on the standard curve chart opens a menu (Figure 6.8). Fig. 6.8. The Standard Curve Chart Menu. This menu includes the following options: • Copy Graph: Copy Graph copies the standard curve chart to the clipboard.
Section 6 Data Analysis Module • Print Data: Print Data prints the Standard Curve/CT Results spreadsheet to your specified printer in Windows • Print Graph: Print Graph prints the graph to your specified printer in Windows • Restore Graph: Restore Graph is active only after you zoom in on the chart. Clicking Restore Graph returns the standard curve chart to its un-zoomed state • Show Labels: Show Labels labels standards and unknowns with the well name as shown in Figure 6.9 Fig. 6.9.
Section 6 Data Analysis Module Fig. 6.10. The Plate Spreadsheet. The spreadsheet displays only one fluorophore at a time. You can use the Plate spreadsheet fluorophore selector buttons found under the Plate spreadsheet to determine which fluorophore is displayed in the spreadsheet. Each well has a colored bar at the top of the cell, which is the color used in the amplification chart to display the amplification trace for this well and fluorophore. The sample type is always displayed in this spreadsheet.
Section 6 Data Analysis Module Columns can be sorted by clicking on the column headings and reordered by clicking and dragging to move columns. Data can be exported by right-clicking on the spreadsheet and selecting Export to Excel. Data can be printed by right-clicking on the spreadsheet and selecting Print. None of the data in this spreadsheet may be edited. 6.4.
Section 6 Data Analysis Module Fig. 6.13. The Set Data Analysis Window. To set the Data Analysis Window: 1. Right-click on the PCR amplification plot, and click Set Data Analysis Window in the menu. 2. Click either the Beginning of Cycle or End of Cycle option to select data from the beginning of the cycle or the end of the cycle, respectively. 3. Set the window width in the Set Window Width scroll box. 4. To use all data points in the cycle, select Use Full Cycle Scan. 5.
Section 6 Data Analysis Module Where: • Oi is the filtered value for a given data point, i • Ri is the unfiltered value for data point i • c is a weighted factor with a value of 2 • M is the arithmetic mean of all data points for the well within the given cycle. The rolling boxcar filter is the arithmetic mean of the data readings i – (w–1), where w is the filter width.
Section 6 Data Analysis Module Start and Ending Cycle Mode To change the baseline range for all wells, click Select All. Then click Edit Range to enter the start and end cycle for baseline calculation (Figure 6.16). To change the baseline for individual wells, click on a well and then click Edit Range to enter the start and end cycle for baseline calculation. Fig. 6.16. The Both Start and Ending Cycle Mode.
Section 6 Data Analysis Module Fig. 6.18. The Ending Cycle Only Mode. Setting the Threshold Manually By default, the iQ5 software automatically calculates the threshold. You can override autocalculation in one of two ways: • On the amplification chart, move the cursor over the displayed threshold line until the cursor icon becomes a hand.
Section 6 Data Analysis Module The alternative to viewing the data in All Candidates mode is to view the data in Single Point mode. Adjust Graph Rescale or change the amplification plot from linear to log, or vice versa, by selecting Adjust Graph. The Chart Axes Range Definition window appears (Figure 6.19). Fig. 6.19. The Chart Axes Range Definition Window.
Section 6 Data Analysis Module Fig. 6.20. The Define Trace Style Window. To modify a trace style: 1. Select the Sample Types Group to be modified (for example, Standards). (Note that green is the default color that is automatically assigned to the Standards group when this button is active.) 2. To specify a different color for a given sample type group on the plate, click on the colored button in the Select Color column. 3. Select the symbol type (the default style is “None”). 4.
Section 6 Data Analysis Module Restore Graph Option Click Restore Graph to redraw the graph to its original size after you zoom in on the graph. Show All Traces Option Click Show All Traces to show all traces after you single out one or more using Display Wells or by clicking on a trace in the amplification plot. 6.5.3 Data Export Options The amplification plot menu offers four additional options for printing and exporting graphs or data.
Section 6 Data Analysis Module Fig. 6.21. Exporting PCR Quant Data Tables to Microsoft Excel. The Export to Excel command is useful for exporting exact values from the spreadsheet. When the Export to Excel command is selected from the menu, an Export to Excel file save box is displayed. Choose a location where the Excel file is to be saved and click Save. The iQ5 software automatically exports the selected data into a protected workbook.
Section 6 Data Analysis Module Fig. 6.22. Melt Curve Chart. The data in the melt peak chart are derived from the melt curve chart (Figure 6.23). Fig. 6.23. Melt Peak Chart. The data in the melt peak chart show the negative rate of change in fluorescence with changing temperature, that is: –d(RFU)/dT Where T is temperature. In analyzing the melt peak data, the software assigns Begin and End temperatures to each peak and then calculates an area beneath that curve.
Section 6 Data Analysis Module The vertical temperature bar may be dragged to any position on the plot. The temperature bar on the melt curve plot moves along to the same position as the temperature bar on the melt peak plot is moved. If you place the cursor over a trace, so that the pointer turns into a hand, the trace will be identified in the small box placed in the top right corner of the plot. The chart can be expanded to fill the window by clicking the + box in the top left corner of the plot.
Section 6 Data Analysis Module Fig. 6.25. A Melt Peak Chart. 6.6.4 Melt Curve/Peak Control Area The control area appears between the spreadsheet and the plots. This area contains buttons to access the Display Wells and Analyze Wells dialog boxes and the Restore Defaults function (Figure 6.26). Fig. 6.26. The Control Area. The position of the temperature bar, threshold bar, and the height and well of the currently selected peak are also displayed here. You cannot edit any of these fields.
Section 6 Data Analysis Module The spreadsheet displays the following information for each peak: • Peak ID: A unique identification number in the format RCC.N, where R is a row letter, CC is a column number, and N is a number beginning with 0, 1, 2, etc. In Figure 6.27, the first peak for well E3 is identified as E03.0 and the second peak as E03.
Section 6 Data Analysis Module A B Fig. 6.28. Allelic Discrimination Plot; RFU View (A) and Threshold Cycle View (b). Genotype assignments for unknown samples are determined by plotting the RFU for one fluorophore (allele 1 on the x-axis) against the RFU for the other fluorophore (allele 2 on the yaxis) on the allelic discrimination plot.
Section 6 Data Analysis Module In Manual Call mode, the plot displays the RFU or threshold cycle data only. The threshold bars do not appear, and modifications to calls are made manually by using the drop-down menu in the Call column of the data spreadsheet. Alternatively, modifications to calls are made by choosing the call type from the radio buttons in the Allele Call box and then clicking and dragging to select desired samples in the graph.
Section 6 Data Analysis Module Fig. 6.30. The Allelic Data Spreadsheet. • In Automatic Call mode, the iQ5 software creates the assignments based on the positions of the vertical and horizontal bars • In Manual Call mode, the Call column becomes editable through a menu.
Section 6 Data Analysis Module more wells on the plot or in the spreadsheet and then return to Automatic Call and the software will position the vertical and horizontal bars based on the new definitions, and then make genotype assignments. 6.7.5 Manual Calls To make manual calls on the plot: 1. Click Manual Call. A new box appears in the window (Figure 6.32). Fig. 6.32. The Manual Call Specification Options Box. 2. Select one of the allelic call types by clicking its radio button. 3.
Section 6 Data Analysis Module Fig. 6.34. The Select Cycle Drop-Down List Box. 6.7.7 Vertical Threshold The Vertical Threshold box (Figure 6.35) displays the current position of the vertical bar. This box is not editable. The positions of the vertical bar along with the position of the horizontal bar determine the genotype assignment for unknown samples. Fig. 6.35. The Vertical Threshold Box. 6.7.8 Horizontal Threshold The Horizontal Threshold box (Figure 6.
Section 6 Data Analysis Module Reference: Livak JL, Marmaro J and Todd JA, Towards fully automated genome-wide polymorphism screening, Nature Genetics, 9, 341-342 (1995) 6.7.10 Restore Default At any time, all modifications to the allelic discrimination data can be reversed by clicking Restore Default. The iQ5 software will reload the original x-axis allelic 1 fluorophore and y-axis allelic 2 fluorophore data, original display mode and Show Labels setting that were originally saved in the .
Section 6 Data Analysis Module • Positives & Negatives: Select this method to use positive and negative controls to define/call unknown samples. Samples are considered positive if they are greater in RFU value than the positive control average minus the tolerance. Samples are considered negative if their RFU value is less than the negative control average plus the tolerance End Point Tolerance End Point Tolerance defines the margins for sorting unknowns as positives or negatives.
Section 6 Data Analysis Module Results The Results box lists the following information: • Source of Data: Displays the analysis mode of the source data. For End Point only data, this is Background Subtracted. For end point analysis of PCR quantification data, the source of data must be either PCR Base Line Subtracted or PCR Base Line Subtracted Curve Fit.
Section 6 Data Analysis Module • Define Controls: Lists any positive or negative controls defined in the original plate setup file. You may also add new controls or edit existing controls in this column by clicking on the drop-down menu on the right side. The options are (+) Positive, (–) Negative, or blank. Alternatively, you may type the letter ‘p’ or the plus sign (+) to select a positive control, and you may also type the letter "n" or the minus sign (–) to select a negative control.
Section 6 Data Analysis Module Gene expression analysis is the most common real-time PCR application. Therefore, this manual uses terminology that is specific to this kind of study. You can safely substitute “gene” with the word “sequence” or “target” when you read about how to use the gene expression module. Without stringently quantified controls, you cannot use this part of the iQ5 software to evaluate target concentrations of particular sequences relative to each other.
Section 6 Data Analysis Module Basic Workflow Steps for Gene Expression Analysis 1. In the PCR Quant screen with a .opd data file open, assess threshold and baseline information for the data file and make changes if necessary. 2. Click the Gene Expr tab. 3. Make any changes to the Gene and Condition (for example, Sample and Treatment) assignments in the gene expression plate interface. 4.
Section 6 Data Analysis Module 3. Make any changes to Gene and Condition (for example, Sample and Treatment) assignments in the gene expression plate interface. • Expand the gene expression plate interface view by clicking on the “+” button to make well identification and selection easier.
Section 6 Data Analysis Module • Number of cells from which nucleic acid was isolated. Rel Quant/number of cells represented in each sample • Mass of tissue from which nucleic acid was isolated. Rel Quant/mass of tissue represented in each sample To calculate Relative Quantity (dCT): 1. In the PCR Quant screen with a .opd data file open, assess threshold and baseline information for the data file and make changes if necessary. 2. Click the Gene Expr tab. 3.
Section 6 Data Analysis Module 1. In the PCR Quant screen, assess threshold and baseline for the data file and then make changes if necessary. 2. Click the Gene Expr tab. 3. Highlight and select the wells to be edited in the gene expression plate interface. NOTE: Expanding the gene expression plate interface view by clicking on the “+” button makes the entire plate area visible for easier well identification and selection. 4.
Section 6 Data Analysis Module • You have a multiplex experiment. In this case all sample types in the varying fluorophores must be the same or; • You are importing multiple .opd (data files) into a Gene Study Make sure the Copy conditions to all data sets button is highlighted (active) when you assign condition (sample) names as shown in Figure 6.40. Active is the default state for this button. Fig. 6.40. Copying Gene and Condition Settings to all Fluorophores.
Section 6 Data Analysis Module 1. Activate the copy command by first selecting the wells to be copied from the plate interface. 2. Right-click on the plate interface to reveal the Copy Gene or Copy Condition options, and select the desired option. 3. Once the Copy Gene or Copy Condition option has been selected, the Paste Gene/Condition Name text from the menu becomes active (no longer grayed out). 4.
Section 6 Data Analysis Module To access the gene list: 1. Make sure the Settings button is activated. If not, click Settings. See the example in Figure 6.42. 2. Click Gene List. The list of genes appears in the spreadsheet directly below the Gene List option button as shown in Figure 6.42. Fig. 6.42. Selecting the Gene List Option. Assigning a Reference Gene To assign a reference gene: 1. With the Settings button active, select Gene List. 2.
Section 6 Data Analysis Module Reaction Efficiency and Gene Expression Analysis Efficiency describes how much of your sequence of interest is being produced with each cycle. An efficiency value of 100% means that you are doubling your sequence of interest with each cycle. People focus on efficiency for a number of reasons.
Section 6 Data Analysis Module Setting Control Conditions You can assign one condition as the control condition by selecting a checkbox next to the sample in the Condition List as shown in Figure 6.44. The iQ5 software assigns the control sample a value of 1 for every gene, and all other conditions will be presented with values relative to this one. This makes it simple to evaluate fold expression relative to the chosen (control) sample. NOTE: The control condition will have a value of 1 for all genes.
Section 6 Data Analysis Module 6.9.5 Applying an Analysis Method 1. Choose an analysis method by clicking one of the option buttons: • Normalized expression (ddCt) is the default and requires you to first choose a reference gene) or • Relative quantity (dCt) 2. Click on Recalculate to apply the new settings. NOTE: Many changes made to the plate setup and analysis options require that you recalculate. If you do not immediately see the change you expect to see, click Recalculate (Figure 6,46). Fig. 6.
Section 6 Data Analysis Module Corrected Values Calculations The efficiency value (E) used in gene expression calculations has an associated error.
Section 6 Data Analysis Module Where n = number of measurements The Standard Error for Normalized Expression equation is shown below: SE Re l Quant sample (Re f 1) SE NFn = NFn × n × Re l Quant sample (Re f 1) Where NF = Normalization Factor 2 SE Re l Quant + n × Re l Quant 2 sample (Re f 2 ) sample (Re f 2 ) SE Re l Quant +L+ n × Re l Quant sample (Re f n ) sample (Re f n ) The Standard Error for Normalized GOI equation is the following: SE GOI norm SE NF
Section 6 Data Analysis Module 6.9.7 Graphing Options for Expression Data Graph Expression Relative to Control or Relative to Zero These options, shown in Figure 6.50, allow you to present data with bars originating at 1 (relative to control) or at zero (relative to zero). If you assign a control in your dataset, selecting the option to graph data relative to control allows you to quickly visualize upregulation and downregulation. The values graphed are exactly the same. Fig. 6.50.
Section 6 Data Analysis Module Scaling Options These options, shown in Figure 6.52, are only active in Normalized Expression mode; they allow you to calculate and present your data in a manner that is best suited for your experiment. Fig. 6.52. Scaling options for Normalized Gene Expression. • Scale to Highest: This option recalculates the normalized expression for each gene by dividing the expression level of each condition by the highest expresser.
Section 6 Data Analysis Module Graph Error Options The default presentation for the error bars is +/– one standard deviation (Figure 6.54). You can change the multiplier to get +/– 2 or 3 standard deviations. Fig. 6.54. Selecting Error Bar options for Gene Expression Analysis. Gene Expression Module Context Menu Right-clicking on the gene expression chart will reveal the context menu shown in Figure 6.55. Fig. 6.55. The Gene Expression Chart Context Menu. Sorting Option Sorting Option (Figure 6.
Section 6 Data Analysis Module arrow buttons to move the member to the desired position within the list of names. More than one Gene or Condition Name can be selected (highlighted) by clicking on the desired names. Note that when the Manual order radio button is active, a total of four arrow buttons are displayed.
Section 6 Data Analysis Module 3. Click Copy Graph. 4. Switch to or open the document into which you will paste the graph. 5. Paste the graph image by choosing Edit, then Paste, or by pressing CTRL+V. 6.9.8 Normalized Expression Calculations The normalized expression calculation is stated below. To see how the iQ5 software calculates relative quantities, go to relative quantity calculations.
Section 6 Data Analysis Module Standard Deviation for the Normalized Expression Rescaling this value is accomplished by dividing the standard deviation of the normalized expression by the normalized expression value for the highest or lowest expresser depending on which scaling option you choose. When a control is assigned you need not perform this rescaling function on the standard deviation as illustrated below.
Section 6 Data Analysis Module Relative Quantity When a Control is Assigned With a control assigned relative quantity (dCT) for any sample for all genes is calculated as follows. Where E = Efficiency of primer (primer/probe) set this efficiency is calculated as follows (% Efficiency * 0.01 + 1) where 100% = 2 CT (Control) = Average CT for the sample which has been assigned as a control CT (Sample) = Average CT for the sample This is where the calculations differ from those outlined by Dr.
Section 6 Data Analysis Module How does normalized expression as calculated by this software compare to the model introduced by Dr. M. Pfaffl, et al. If you only evaluate one reference gene and one gene of interest you will get exactly the same results using the iQ5 software as you would using the model introduced in this paper. Standard deviations may be slightly different. How does normalized expression as calculated by this software compare to the model outlined by Dr.
Section 6 Data Analysis Module performed by activating the Enable for Gene Study button found in the Gene Expression window of the Data Analysis module (Figure 6.57). Fig. 6.57. The Enable for Gene Study button is located above the plate interface section of the Gene Expr tab window. When a file has been saved with the Enable for Gene Study button active, it allows the saved file to be added to a Gene Study. The default setting is that this button is not enabled.
Section 6 Data Analysis Module 6.10.2 Creating a Gene Study File A Gene Study file must contain data from at least two different .opd files. The process of creating a Gene Study file is a 2-step process. STEP 1: Prepare .opd files for gene expression analysis. 1. In the PCR Quant tab, establish the desired experimental conditions, such as baseline and threshold and the wells to be included in the analysis. NOTE: Additional wells can be excluded or re-included later from the gene expression plate interface.
Section 6 Data Analysis Module • Created Date: This column displays the creation dates of the .opd files to be included in the Gene Study. Note that these dates are NOT the dates of the most recent save event for the listed files. 6.10.3 Gene Expression Study Manager The columns and rows of the Gene Expression Study Manager (Figure 6.59) are adjustable by clicking, holding and dragging the column/row separator lines.
Section 6 Data Analysis Module corresponding row. If one or more files have been selected, clicking the Remove button will remove those file(s) from the Gene Study. The Notes field is also editable from the Gene Expression Study Manager. Once the desired changes have been made, click OK to close the Gene Expression Study Manager and return to analysis of the Gene Study file in the gene expression module. Remember to immediately save your edits to the Gene Study before continuing with your analysis.
Section 6 Data Analysis Module dominant inter-run calibrator. The average dCT value of the dominant inter-run calibrator will be used to adjust all CT values within the Gene Study. To find the dominant inter-run calibrator, the iQ5 Inter-run Calibration algorithm first calculates the average of the dCT values for all inter-run calibrators of a given gene. The iQ5 software uses a multi-tiered algorithm to determine the dominant inter-run calibrator.
Section 6 Data Analysis Module Details of the Inter-run Calibration calculations can be accessed by clicking the Inter-run Calibration button. The resulting Inter-run Calibration window (Figure 6.61) will display the comparative fluorophore calculations per gene (pair-wise comparisons). • You can choose to view the inter-run calibrator calculations for different genes in your assay by selecting a Gene Name from the Select Gene drop-down menu.
Section 6 Data Analysis Module For post-run editing of the Plate Setup saved in a data file: 1. From the Workshop module: a. Click Data File above the directory of the home workshop. Navigate the directory until the desired data file is found. Double-click the file name to bring the file directly into the Data Analysis module. or b. Click Data File above the directory of the home workshop. Navigate the directory until the desired data file is found.
Section 6 Data Analysis Module 9. Select the type of replicate loading desired. 10. Click or drag across the plate to define wells with the selected fluorophore and sample type. 11. Continue defining the remaining wells that will contain the first fluorophore by changing to any other sample type icons required.
Section 6 Data Analysis Module 6.12.1 Report Viewer Options The following options are available from within the Report Viewer window: • Select Report: Use this drop-down menu to select the level of detail in the report for your dataset. The number and type of reports available will vary according to the Data Analysis Module currently being used • Sort Data By: Within a given report, all data displayed in tables can be sorted by the parameters listed in the drop-down menu.
Section 6 Data Analysis Module • PCR Quant Detailed: This report template includes all of the data available in the PCR Quant module of the iQ5 software – including the PCR amplification chart, CT value spreadsheet, and analysis parameters. If the dataset being analyzed also contains a standard curve, the standard curve chart and spreadsheet data will also be included in this report.
Section 6 Data Analysis Module parameters. In addition, if all fluorophores have been selected for display in the PCR Quant module, the PCR Amplification Chart and PCR Baseline Analysis Parameters will also be displayed. • Gene Expression Graph only: This report template displays only the Gene Expression chart, exactly as analyzed and formatted within the gene expression module.
Section 7 Calibrating the Instrument Section 7. Calibrating the Instrument This section contains information on the following topics: o Calibration overview (page 111) o Components required for calibration (page 111) o Preparing calibration plates (page 112) o Performing calibrations (page 112) o Viewing calibration files (page 119) o Troubleshooting optics with the Image Window (page 120) 7.
Section 7 Calibrating the Instrument The following reagents and accessories are required to calibrate the iQ5 instrument: • iCycler iQ calibrator dye solution kit (catalog #170-8792), which contains iQ5-specific pure dye solutions and external well factor solution • 3 x 96-well PCR plates or preferred reaction vessel • Optical-quality sealing tape or preferred sealing method NOTE: Calibrator dye solutions are NOT required for calibrating the MyiQ system. 7.
Section 7 Calibrating the Instrument Calibrations are performed in the order as they appear in the tabs of the Calibration module: • Mask Alignment • Background Calibration • Persistent Well Factor Generation • Pure Dye Calibration 7.4.1 Performing Mask Alignment Before you align the mask, complete the following steps: 1. Prepare an external well factor plate (see section 7.3) 2. Place the external well factor plate in the iCycler thermal cycler. 3.
Section 7 Calibrating the Instrument 3. Click Take an Exposure. The Image screen displays the fluorescence detected by the CCD camera for each of the 96 wells on the plate. Pixels that are saturated will be shown in pink 4. Click Show Mask. The Mask is displayed as an array of green boxes (Figure 7.2). Fig. 7.2. Camera Image with Overlying Mask Array. 5. If any pink (saturated) pixels are present in the mask, reduce the exposure time and retake an exposure.
Section 7 Calibrating the Instrument 3. Place the background calibration plate in the iCycler thermal cycler. 4. Select the Background tab in the Calibration module. The Background window is shown in Figure 7.3. Fig. 7.3 The Background Window. To perform background calibration: 1. Select a well sealing type (film or caps) from the drop-down list. 2. Select a vessel type (plates or tubes) from the drop-down list. 3. Click Collect Background Readings. 4.
Section 7 Calibrating the Instrument To generate persistent well factors: 1. Select a well sealing type (film or caps) from the drop-down list. 2. Select a vessel type (plates or tubes) from the drop-down list. 3. Click Collect Persistent Well Factors. 4. When the iQ5 system completes the persistent well factor calibration run, a dialog box appears with the message, "Persistent Well Factor Calibration Run Complete". 5. Click OK to exit.
Section 7 Calibrating the Instrument To perform a pure dye calibration run: 1. Select from the Pure Dye Plate Setup file selector window or click Create New. Ensure appropriate dyes are selected that are compatible with the filter setup of your connected instrument. If incompatible dyes are selected the following error will appear. 2. Click Run Pure Dye Calibration. 3. When the iQ5 software completes the pure dye calibration run, a dialog box appears with the message, "Pure Dye Calibration Run Complete".
Section 7 Calibrating the Instrument 7.4.5 Editing and Creating Pure Dye Plate Setups Selecting a Pure Dye Plate Setup Select a Pure Dye Plate Setup from the file selector window within the Pure Dye Calibration tab. When loaded, the fluorophores on the plate will display within the plate display in the lower righthand corner of the Pure Dye Calibration tab. Check the Seal Type and Vessel Type fields.
Section 7 Calibrating the Instrument Select/Add Fluorophores To choose the fluorophores needed to calibrate the instrument, select from the predefined fluorophores or add custom fluorophores by clicking Select/Add Fluorophores. The Fluor Selector will be displayed (Figure 7.8). Fig. 7.8 The Select/Add Fluorophores Dialog Box Add a fluorophore from the list displayed by clicking the checkbox next to the fluorophore name.
Section 7 Calibrating the Instrument To determine what fluorophore dyes an instrument has been calibrated for within the iQ5 software, select Calibration Data from the View menu (Figure 7.9) to open the Calibration Data file view. Fluorophore dyes for which the instrument has been calibrated will be listed by name in the summary table. Fig. 7.9. The View Menu. Similarly, the contents of the background and external well factor calibration files can also be accessed in this manner. 7.
Section 7 Calibrating the Instrument The Camera Status screen (Figure 7.11) provides feedback about the camera.
Section 8 User Profiles Section 8. User Profiles This section contains information on the following topics: o User Preferences (page 122) o User Administration (adding and deleting users) (page 128) o Logging on (page 130) o Switching Users (page 131) o Changing Password (page 131) o Defining Roles (page 132) To assist with the management of files and data created by the iQ5 software, one or more user profiles may be created for users of the MyiQ2, MyiQ or iQ5 systems.
Section 8 User Profiles • Allelic Discrimination module analysis settings • End Point module analysis settings • Gene expression module analysis settings 8.1.1 File Paths Preferences Each file created by the iQ5 software is destined to a specific folder location on your computer, this is known as a “file path”. The File Paths preferences box (Figure 8.1) can be used to select the folders that you wish to save your files into when logged into the iQ5 software.
Section 8 User Profiles 8.1.2 Plate Setup Preferences The Plate Setup preferences box (Figure 8.3) can be used to define the default conditions when a user creates a new plate setup. Fig. 8.3. Plate Setup Preferences.
Section 8 User Profiles IMPORTANT NOTE: The Fluorophores preference box will display all fluorophores available to the MyiQ2, MyiQ and iQ5 systems. If both dyes that can and cannot be detected by the connected instrument are selected in user preferences, only fluorophores that are compatible with the connected instrument will be displayed. If only fluorophores that are not available for detection by the connected instrument are selected, by default the Plate Setup window will display FAM only. 8.1.
Section 8 User Profiles • Last File Used: This option will select the last Protocol, Plate Setup, Run Set or Data File used as the file to display in the Workshop screen when the iQ5 software starts up • Data File: If you wish to have a specific data file selected when the iQ5 software opens, select Data File and then use the browse button to navigate the data file that you wish to have selected on application startup • Run Set: If you wish to have a specific Run Set file selected when the iQ5 softwar
Section 8 User Profiles 8.1.6 Allelic Discrimination Module Preferences The Allelic Discrimination preference box (Figure 8.7) can be used to set the default display conditions used in the Allelic Discrimination screen. The Display Mode can be set to Threshold Cycle or RFU using the radio buttons. The radio buttons for the Normalize Data option become active only when the Display Mode is set to RFU. Fig. 8.7. Defining Allelic Discrimination Display Options. 8.1.
Section 8 User Profiles 8.1.8 Gene Expression Module Preferences The Gene Expression module preference box (Figure 8.9) allows you to predetermine several analysis and display options associated with analyzing CT values for gene expression analysis. Fig. 8.9 Defining Analysis and Display Options for the Gene Expression Module. • Relative to: This option allows you to present data with axes originating at 1 (relative to control) or at zero (relative to zero).
Section 8 User Profiles 8.2.1 Adding New Users Users are added using the Defining Users spreadsheet (Figure 8.10). Fig. 8.10. The Defining Users interface of the User Profile Module. • User Name (REQUIRED): The User Name is a set of alphanumeric characters that uniquely defines each user. It can be up to 15 characters long and composed of upper and/or lower case characters.
Section 8 User Profiles Adding a user will create a folder with the new user’s name. This folder will be created in the BioRad/iQ5/Users folder and will be the default folder for all files saved when the user is logged into the software. 8.2.2 Deleting Existing Users Users are removed using the Defining Users spreadsheet. 1. In the Defining Users spreadsheet, check the box in the Delete column for the user that is to be removed. 2. Click Save User Changes.
Section 8 User Profiles 3. Click OK and enter the correct information. If you have forgotten your password, it may be reset by the iQ5 Administrator. 4. The current user can be determined by looking at title bar (in the top-right corner) of the software. The current user name will be displayed in parentheses next to the Bio-Rad iQ5 header (Figure 8.13). Fig. 8.13. User Name Displayed in the iQ5 Software Title Bar. 8.3.1 Switching Users To switch a user: 1.
Section 8 User Profiles 8.3.3 Defining Roles Fig. 8.15. The Defining Roles interface of the User Profile module. Figure 8.15 lists the set of features and functions (permission) that can be granted by the iQ5 Administrator to users assigned to one of three non-administrator roles. The Administrator can customize the roles of a Principal, Operator, and Guest by checking or unchecking the box associated with each function and role. To apply the selected changes, click Save Role Changes.
Section 9 Instrument Maintenance Section 9. Instrument Maintenance This section contains information on the following topics: o Cleaning the system (page 133) o Recommended fluorophores and filter specifications (page 133) o Accessing and cleaning the filters (page 134) o Replacing the lamp (page 135) 9.1 Cleaning the Real-Time PCR Detection System Take care not to spill liquids onto or into the iCycler thermal cycler or the optical module.
Section 9 Instrument Maintenance • Filter position 4 545/30X 585/20M TAMRA, Cy3 • Filter position 5 575/30X 625/30M Texas Red, ROX • Filter position 6 630/30X 685/30M Cy5, LC640 NOTE: The filter designation 485/30X indicates that this filter will allow light between 475 and 595 nm to pass through. The first number, 485, indicates the center of the wavelength of light. The second number, 30, indicates the total breadth of wavelengths of light that can pass through it.
Section 9 Instrument Maintenance You may clean the optical filters by wiping them gently with lens paper and 70% ethanol. You may obtain lens paper and lens cleaning solution by ordering a Lens Cleaning Kit from Bio-Rad (catalog #170-7731). After cleaning both sides of an optical filter, hold it up to the light and make sure that no smudges, fingerprints, debris, or water marks remain. Once cleaned, reinsert the optical filters as described above. 9.
Appendices Appendix A: Warranty The MyiQ2, MyiQ and iQ5 Real-Time PCR Detection Systems are warranted against defects in materials and workmanship. For specific warranty information, contact your local Bio-Rad office. If any defects should occur during the warranty period, Bio-Rad will replace the defective parts without charge. However, damage or defects resulting from any of the following causes are specifically excluded: 1. Improper operation. 2. Use of improper solvent or sample. 3.
Appendices Appendix B: Troubleshooting Error Messages Cause: Selecting or creating plate setup files that contain fluorophores not compatible with the connected instrument. The warning message above will be displayed at the bottom of the screen. This message indicates that while the plate setup can be viewed and edited, the plate setup contains fluorophores that cannot be used to collect new data. Solution: If you wish to view or edit the plate setup, no corrective action is required.
Appendices Cause: This error appears when the software/computer is unable to communicate with the camera. The iQ5 software and camera are on but the iCycler base unit is powered off. Solution: Close the software, turn the camera on and restart the iQ5 software. Check that the USB cable is securely connected to both the camera and computer. Ensure that the 2.0 high-speed enabled USB port on the computer is being used and all hibernate and power save settings are disabled. Refer to section 1.2.5 and 1.
Appendices Cause: Well factor data not found. This error occurs when a run start is attempted using a vessel and seal combination the instrument has not been calibrated for. Solution: Well Factors vessel and seal type for your plate must match collected Persistent Well Factor calibration files. The instrument should ideally be re-calibrated for the desired vessel and seal type combination.
Appendices Cause: Bad protocol file. There is no step defined for data collection or analysis in the thermal protocol. Solution: At least one data acquisition step must be present in the thermal protocol. Open the protocol editor and specify a data collection at one step of the protocol. A data collection step is indicated by a yellow (real-time data collection) or green (melt curve data collection) camera icon at the appropriate step in the graphical display of the protocol. Refer to section 4.
Appendices Cause: This is not an error message but a warning reminder that the iQ5 software is running in emulation mode. In emulation mode the camera is not detecting and collecting data from the plate and is generating emulated run data for demonstration purposes only. Solution: If you wish the camera to detect and collect data from your experimental plate, exit the software. From the Start Menu, select programs > Bio-Rad > iQ5 >iQ5 to restart the software in standard data collection mode.
Appendices Appendix C: Product Ordering Information Catalog Number Product Description 170-9790 MyiQ2 Real-Time PCR System, includes iCycler thermal cycler, 96-well optical reaction block, MyiQ2 optics module, iQ5 system software on CD-ROM, optical-quality 96-well PCR plates, sealing tape, communication cables, system accessories, power cords, instructions 223-9441 96-well Thin Wall PCR Plates, 25 per box HSS-9601 Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plates MSB-1001 Optical Quality Sealing
Bio-Rad Laboratories, Inc. Web site www.bio-rad.
