PrimeQ OPERATOR’S MANUAL Version 5.
How to use this guide Please read all the information in this guide before using the unit. Le rogamos lea cuidadosamente la información contenida en este folleto antes de manipular el aparato. Veuillez lire attentivement toutes les instructions de ce document avant d’utiliser l’appareil. This operator guide provides the basic information to get you started on the PrimeQ real-time PCR system.
Chapter 6 Troubleshooting and Glossary Identifying problems with the instrument and/or the PCR Glossary of terms used in real-time PCR 4
Contents 1 Introduction............................................................................................................................... 13 1.1 PrimeQ ............................................................................................................................... 15 1.1.1 PrimeQ features ......................................................................................................... 15 1.1.2 The thermal system ......................................................
1.16 Installing the filter cartridges .......................................................................................... 31 1.16.1 The role of the filter cartridge ..................................................................................... 31 1.16.2 Filter cartridge care instructions ................................................................................ 32 1.16.3 Filter cartridge installation .................................................................................
3.3 3.3.1 Home page ................................................................................................................ 55 3.3.2 Main screen ............................................................................................................... 56 3.3.3 Navigation bar ............................................................................................................ 56 3.3.4 Title bar functions ............................................................................
4.3.2 PrimeQ Report ......................................................................................................... 112 4.4 Passive reference dye (PRD) correction .......................................................................... 113 4.5 Analysis method: Baseline correction .............................................................................. 114 4.5.1 Assay requirements .................................................................................................
4.10.5 4.11 Multiplex assays ........................................................................................................... 162 4.11.1 5 Multiplex setup ......................................................................................................... 162 Technical information ............................................................................................................. 165 5.1 Operator maintenance ......................................................................
1 Introduction Safety and installation information About this chapter This chapter provides information on general safety aspects, definitions, advice and instructions for unpacking and installing your instrument. It also gives general information about the instrument and the control software, system requirements, basic procedures, control mechanisms and software installation.
1.1 PrimeQ PrimeQ - the real-time nucleic acid detection system from Techne - has been designed with the advantage of an open chemistry format that allows the end user full flexibility in the methods and research they wish to pursue. This is complimented by the user-friendly application software, Quansoft, for easy setup of experiments and rapid analysis of data. 1.1.
1.2.3 Program Editor Individual cycle steps, stages or temperature ramps can be added quickly and easily together with other parameters to build and display the thermal cycling program. 1.2.4 PrimeQ reports Comprehensive, completely user-customizable reports can be generated from any of the analysis ® methods. All result types including graphs can be exported to Microsoft Word, PowerPoint or other programs currently used for publication purposes. 1.2.
1.3 Unpacking When unpacking the unit, check that the following have been removed from the packing: • This PrimeQ operator guide • A CD containing an electronic copy of this guide and Quansoft software • The PrimeQ unit including block • Filter cartridges in box • Mains lead(s) • Guarantee card • Decontamination certificate • USB cable Keep the original packaging in case you ever need to return the unit for service or repair.
1.7 Thermal seal The specification given in this Guide is based on the use of an optical heat seal. Other types of optical seal may be used; however any seal must be able to withstand the temperatures you are using without any danger of deforming to the point where it splits. The user should check the suitability of the seal to be used to ensure there is minimal sample evaporation. Where applicable, you should use a heat sealer which seals at a temperature of approximately 170ºC.
The unit must be connected to a PC as described in section 1.14. 1.9.2 Installing the instrument 1.9.2.1 Warning HIGH TEMPERATURES ARE DANGEROUS: they can cause serious burns to operators and ignite combustible material.
1.9.3 Operator safety All users of Techne equipment must have available the relevant literature needed to ensure their safety. It is important that only suitably trained personnel operate this equipment in accordance with the instructions contained in this Guide and with general safety standards and procedures. If the equipment is used in a manner not specified by Techne, the protection provided by the equipment to the user may be impaired.
The fused plug supplied with the mains cable is fitted with a 10 amp fuse to protect the instrument and the operator. The rating plate is on the rear of the unit. Note: the unit can work in the range 100V to 230V. 3. Plug the mains cable into the socket on the rear of the unit. 4. Place the unit on a suitable bench or flat workspace, or in a fume cupboard if required, ensuring that the air inlet vents on the underside are free from obstruction. 5.
Le fusible (10A) à l’intérieur de l’appareil est destiné à assurer la protection de l’appareil et de l’opérateur. Les appareils fonctionner sur 100V et 230V La plaque d’identification se trouve à l’arrière de l’appareil. 3. Raccordez le câble d’alimentation à la prise située à l’arrière de l’appareil. 4. Placez l’appareil sur un plan de travail ou surface plane, ou le cas échéant, dans une hotte d’aspiration, en s’assurant que les trous d’aération situés sous l’appareil ne sont pas obstrués. 5.
1.10 Before switching on 1.10.1 PrimeQ front view 1.10.2 Fuses Ensure that the correct fuses are fitted for your voltage supply. Fuses should only be changed by a competent person with training if necessary. For voltages from 110 V to 130 V, use T10A fuses only. 1.10.3 Connections on the back of the unit There are two cable connections: the mains power switch and the USB socket located on the rear of the PrimeQ. A. USB computer connection.
1.11 Technical Specification Thermal cycler Block Format: 96 x 0.2ml well low-profile micro tube plate Block Specification: 8 x Peltier block employing quad circuit technology to enhance performance Block Uniformity at 50ºC: < ± 0.25ºC Maximum Heating Ramp Rate: 2.
Computer requirements (Not Supplied) The following are the recommended minimum PC specifications required for running PrimeQ: CPU: Core 2 Duo or equivalent Memory: 2Gb DDR RAM as a minimum Storage: 40Gb DMA hard drive Display: 1024 x 768 resolution minimum, 17 inch digital monitor recommended.
Within these periods we undertake to supply replacements free of charge for parts which may on examination prove to be defective, provided that the defect is not the result of misuse, accident or negligence. On all correspondence, please quote the Serial Number in full and/or the Sales Order Number.
1.14 Installing the application software Always set your PC or laptop from which you are running PrimeQ so that standby is switched OFF. This can easily be achieved through the control panel or: • Right click on the desktop and select Properties. • Select the Screen Saver tab and click on the Power button. • Activate the power scheme entitled Always On from the drop down menu and click Apply. You may need to restart your computer. 1.14.1 Set the decimal symbol to‘.
1.14.2 Loading Quansoft onto the PC To install the Quansoft software onto the PC, follow these five basic steps: 1. Turn on the PC and log on as a user having administrator rights (to be able to install software). 2. Insert the Quansoft CD into the CD-ROM drive and wait for the auto-run function to automatically start the setup (should auto-run fail to start, then the CD must be opened from Explorer and the setup program (Setup.exe) initiated manually by double-clicking). 3.
The unlock code can be obtained in one of two ways: 1. E-mail: PrimeQhelp@bibby-scientific.com.Send an email complete with details of your name, company or institution, mailing address, e-mail address, telephone number, instrument and block serial number (if you have a PrimeQ) together with the unique registration number. 2. Phone: Provide us with your details and registration number by telephoning: +44 (0)1785 810433. A voicemail service is available outside UK office hours.
1.15 Using the LCD control panel 1.15.1 Function keys PrimeQ is controlled primarily from the PC using the application software, although some basic functions can be performed via the LCD screen located on the front of the instrument. The display indicates the instrument name, current status and basic information about any experiment currently running such as its progression. The keypad has three function buttons: Start/Pause: Starts the run or pauses if already running. Stop: Stops the current run.
1.15.4 Before you start Check that: 1. All cables are connected between the instrument and PC. 2. All power cables are plugged in and comply with safety standards. 3. The Quansoft software has been installed on the PC. 1.15.5 Start-up procedure 1. Turn on the power to PrimeQ by pressing the switch at the back of the instrument. A welcome beep can be heard. 2. Turn on the PC and wait for Windows to boot up. 3. Log in under your user name. 4.
1.16.2 Filter cartridge care instructions • The filter cartridges must be handled with special care. • Only lift them from the foam in the box by the sides of the cartridges. • Avoid touching the filters. Loose particles should be removed with a bulb puffer or filtered, pressurized air cleaner. If necessary, gently wipe the surface using 100% alcohol and optical wipes; use a clean optical wipe each cleaning motion. 1.16.
• Excitation/emission wavelengths: As defined in the Add Cartridge settings box. • Dye name: Each cartridge can be assigned up to four different dye names of the user’s choice. 1.16.4 Adding a filter cartridge • In the Access and Editing screen of the Filter Wizard, click the Add button next to the appropriate filter. The Add Cartridge screen will appear. • Set the wavelengths and bandwidths for the excitation and emission filters. This information can be found on the side of the filter cartridge.
1.16.5 Assigning filter descriptions in Quansoft Once the cartridge has been fitted into the cartridge carousel, the system will check for the presence of a new cartridge and then ask the user for the Dye Names. Up to four names can be assigned to any given filter cartridge and the colour coding can be changed by clicking on the Colour icon. The colour chosen here will be the colour of the dot representing the read points on the thermal profile graph.
A message will appear informing the user to Please wait – checking cartridge status. If the removal was performed correctly, the Cartridge Access and Editing table will be updated to display “Not Fitted” next to the slot from which the cartridge was removed. The table below gives details of the PrimeQ filter cartridges.
• On the instrument screen, click on Change, type in the new name and then click OK. Once the name has been changed you need to switch off the instrument before the new name can appear on the LCD screen and take effect. The maintenance screen also gives instrument-specific details including serial number, unique ID and block cycle count. It also shows block constant (calibration) settings.
1.18 LED power settings The LED light source has variable power settings. This prevents PMT blinding and brings fluorescence into its linear range for certain applications. The role of this option is to cut down excess light from the LED source in high fluorescence applications. The three power options are: Low (50mA), Medium (100mA), and High (200mA).
1.20 After use When the samples have finished thermal cycling, remember that parts of the unit, such as the tubes, blocks and associated accessories, may be very hot. Take the precautions listed earlier. Después de su uso Cuando haya finalizado el calentamiento de muestras, recuerde que las piezas del equipo, tales como tubos, bloques y demás accesorios, pueden estar muy calientes. Tome las precauciones mencionadas anteriormente.
2 Getting started Introduction to PrimeQ and real-time PCR About this chapter This chapter gives a description of PrimeQ together with a basic introduction to real-time PCR. It also provides examples of some of the fluorescent chemistries that can be used with PrimeQ.
2.1 Introduction to PrimeQ 2.1.1 Principle of a real-time PCR instrument A real-time polymerase chain reaction (PCR) system performs three main functions: • Cycles the PCR reagents through the specified temperature profile using a specially designed thermal block. • Excites the fluorescent dye at the appropriate wavelength and at the appropriate point(s) in the PCR program.
2.2 Introduction to Real-time PCR 2.2.1 PCR PCR is a powerful biochemical technique that has revolutionised biological research by allowing minute amounts of DNA to be amplified millions of times in just a few hours. PCR allows the selective amplification of a ‘target’ region of DNA lying between two specific DNA sequences (primers). The DNA sequence lying between these primers does not need to be known, therefore PCR allows researchers to amplify target DNA with relative ease and reproducibility.
PCR (qPCR) extends the usefulness of the technology by permitting the reliable determination of starting DNA template. 2.2.3 Real-time PCR on PrimeQ Real-time PCR overcomes the limitations associated with the traditional methods by using fluorescence labelling in conjunction with specialized amplification and detection systems to quantify the amount of product being amplified during the PCR process.
dye is excited at the appropriate wavelength should be proportional to the quantity of target DNA present. For each type of fluorescent chemistry, the fluorophore will emit a fluorescent light that is characteristic of the fluorophore used. A range of spectrally distinct fluorophores are commercially available, introducing the possibility of quantifying multiple targets with different probes in the same reaction well, otherwise known as multiplexing. 2.3.
fluorescence signal or fluorescence at a longer wavelength which is not detected. The 5’ fluorophore is often called the reporter. Mode of action of hydrolysis probes: During the PCR, as the DNA polymerase extends the upstream (forward) primer, it encounters the bound probe. The 5’ to 3’ exonuclease activity of the polymerase cleaves the probe, releasing the fluorophore into solution, where it is able to fluoresce. The probe is blocked at the 3’ end to prevent extension by the polymerase.
2.4 System overview PrimeQ is a real-time PCR unit into which the operator places a 96-well plate containing the samples to be analysed. The unit is connected to a PC via the USB port on which the application software (Quansoft) is run. The software allows the user to: 1. Create a PCR program defining dyes, read points, thermal cycling and analysis methods. 2. Run the program. 3. Analyse the fluorescence readings.
2.5.1 The optical light path Excitation light from the solid state white light source (1) passes through a light path and enters the filter cartridge. Only light at the desired wavelength is allowed to pass through the excitation filter (2). The light is then reflected through 90° by a dichroic mirror (3) and passed into a fibre optic bundle (4). From there, the light is directed into the wells of the sample plate by the fibre optic bundle, which excites the fluorophore present specific to that wavelength.
PrimeQ’s thermal cycler block accommodates a low-profile 96-well plate. Each block is internally calibrated so that if the block is replaced, the temperature performance is unchanged without having to recalibrate the instrument The thermal block is accessed by opening the PrimeQ sample drawer. This provides the user with access to the block so that plates can be loaded or unloaded. 2.7 Scanning module The scanning module delivers excitation light to the sample wells and then collects the emitted light.
3 Quansoft Using Quansoft About this chapter Accompanying PrimeQ is the intuitive, wizard-based software, Quansoft. This chapter discusses the concepts behind this software and looks at its application in the setting up and running of a realtime PCR experiment. Read the first section for an overview of Quansoft in general terms and a brief discussion of how the various parts fit together. The next section helps the user to navigate around the software and reviews its basic functions and commands.
3.1 Introducing Quansoft PrimeQ is a real-time-PCR unit containing sophisticated thermal cycling and fluorescence detection technology. The user interacts with the system via Quansoft, the user-friendly Windowsbased application software installed on a PC connected to the unit. The intuitive software allows the user to perform three main functions: 1. Create a PCR program and define fluorescent reading points. 2. Run the program. 3. Detect and analyse the fluorescence readings.
Plate Layout Editor Within a matter of seconds, a 96-well PCR plate can be assigned with various sample types, including blanks, controls, standards or user-defined samples, all of which are colourcoded for ease of identification. Results Editor Results for each stage of the program containing fluorescent readings are shown on individual tabs and can be analysed separately using various analysis methods. 3.2 Software overview The diagram below outlines the structure of Quansoft.
3.2.1 Home page As shown in the schematic diagram above, the Quansoft Home page is the starting point for an experiment, providing quick links to all of Quansoft’s most important features. Direct links into the different editor functions allow the user to quickly create a new experiment, re-run a previous experiment, edit a program or to analyse new or existing data. Basic maintenance and filter changing functions can also be performed from links on the Home page. 3.2.
Quansoft’s Plate Layout Editor is a flexible platform for defining a plate layout. In this editor, it is possible to set the name and value of samples used for a standard curve. When placing standards in the layout, the user will be asked to fill in the value of each standard in the Well Information table. To assign units to these standards, such as copies/well or ng DNA, options are available in the drop-down list, or a custom unit can be defined.
Quansoft’s Results Editor displays the results of the run, which can be sent to report or re-analysed with different parameters. 3.3 Using Quansoft 3.3.1 Home page The Quansoft Home page acts as a gateway to the software functions, providing quick and easy links to both the Editors and their associated libraries. Other functions, such as basic instrument maintenance, filter changing, and administrative tasks, can also be carried out from here.
3.3.2 Main screen Shortcuts for experiment setup and data analysis: Run an experiment: goes to the Experiment folder to select an experiment. Create a new experiment: goes to the Experiment Editor from where the user can create a new experiment or edit an existing one. Create a new program: goes to the Program Editor from where the user can create a new program or edit an existing one.
Clicking the Security icon allows customizing of the supervisor password (required for the administrative functions which are accessed under the Instrument icon). Clicking the Instrument icon will allow the user to view instrument-specific details including, for example, the instrument serial number and block cycle count. The default supervisor password is techne - we suggest that you change this as soon as possible.
The Up button allows the user to navigate back to a previous folder (used if the directory structure has been turned off). The View button will change the way the files are displayed in the file view pane (detail, large/small icon, list views). The following buttons are displayed on the Title bar when using one of Quansoft’s Editors: The Finish button (Results Editor) saves the changes made and closes the editor.
View: Allows the user to change the way the files appear in the file view. Choose to display simply as icons or display with further details such as file name and size. Tools: Provides direct access into the editor of choice simply click on the editor name. The Change Quansoft folder… command allows the user to change the destination of any saved files. Quansoft will automatically save all files to My Documents\Quansoft; change the destination here if required. Help: Off- and on-line resources.
3.4 Setting up an experiment 3.4.1 An Overview Schematic diagram showing the options available for setting up an experiment on PrimeQ. The icons in the blue boxes represent the shortcut buttons available from the Home page. START 1: Choose to set up an entirely new experiment in the Experiment Editor, browse or edit previously saved plate layout or program templates or else access the Plate Layout and Program Editors direct from the Home page.
3.4.2 Creating a new experiment • From the Home page click on Create a New Experiment. A blank experiment template will open in the Experiment Editor. The Experiment Editor allows the user to perform three important functions: • Define a program, • Define a plate layout • Define an analysis method Note: only the setting of a program is essential for a run to be performed; plate layout and analysis method can both be defined after the run is complete.
2. Plate Layout Setup: a. From New b. Browse an existing template. c. Edit the selected template 3. Analysis Method Selection This chapter will look at each of these functions in turn. 3.4.3 Setting up a program The PCR program is defined using the top-left area of the Experiment Editor main screen: There are three ways to define a PCR program: Click this button when the program is to be defined entirely from new. Click Browse to search the Program library files for existing templates.
• Run information: The user can enter a user name and add any other comments relevant to the program. • Instrument settings: User-defined settings for heated lid temperature, disabling of the instrument buttons, plate type, sample volume and whether to read entire plate or include a final hold in the PCR program. • Program file view: Displays the structure of the program thereby providing the user with a clear view of the overall protocol.
• Heated Lid: Default 105°C. Set a temperature (possible temperature range from 100°C to 115°C). • Wait For…: Set a ‘wait for’ time (i.e. the time to wait while the lid heats up before the block thermal program begins, hrs:min:sec). Or click the button to wait until heated, the program will then start as soon as the lid reaches the set temperature. • Read Whole Plate: Default ON. Click to Read only filled wells.
The default name New Stage can be changed if required. When a stage name is highlighted in the program file view, the stage parameters box in the centre of the screen allows the general parameters for that stage to be defined. • Heated Lid: The default is to keep the heated lid temperature unchanged. To change, click the button and a time and temperature setting box will appear adjacent (possible temperature range from 100°C to 115°C).
• Temperature: Set using the sliding thermometer or type a value in the box. • Hold Time: Shown in hrs:min:sec up to a maximum of 99:59:59 (minimum 00:00:01). The default setting is seconds, therefore for a time in seconds, type in the number of seconds and press the enter key. To set the time in hours, add “h” after the numerical value, e.g. 1h, and for minutes add “m”. • Number of Reads: Displays the number of readings programmed for the step (maximum of 4).
change between these options. If the user is creating a new protocol and the instrument is not connected, it is not possible to select filter cartridges (the drop-down box is greyed-out). The user will be prompted to choose the filters on connection to the instrument or when trying to run the program. • Light Intensity: Choose low, medium or high depending on the type of fluorophore in use and its concentration. Default: Medium.
• Click Add Ramp to open the ramp read parameters box: • Start Temperature: Starting temperature of the ramp can be anywhere from 4°C. • End Temperature: The temperature can be set to ramp to any temperature up to 98°C. • Temperature Increment: The temperature can be increased in increments of between 0.1 and 5°C. The default is 0.5°C. • Hold Time: The user can choose run the ramp with a set hold at each temperature; this can be any time up to a maximum of 99:59:59 hrs:min:sec. The default is 10 seconds.
The read appears as a separate tab labelled according to the dye name. The temperature profile plot shows the reads as colour-coded points. Once the program is complete, clicking on the program name at the top level in the program file view will show the complete thermal plot: Any parts of the program, such as temperatures, hold times, read points, filters etc. can be edited at any point prior to the run. • Click on Save to save the program to the Program Library.
• Highlight a folder and click Open to open up the program file in Experiment Editor. The template is ready to use. 3.4.3.8 Edit a program A program already present in the Experiment Editor can be edited simply and easily by clicking the Edit button in the Program pane of the Experiment Editor. Clicking the Edit button will open up the program within the Program Editor thus providing all the same functionalities as if defining a program from new.
• On the menu bar in Program Editor, select File and then Save As… • Change the file name if required. The file (.qprg format) will automatically be saved to the directory My Documents\Quansoft\Programs. Change the destination by browsing the file directories shown in the Save in: drop-down menu. The library files can be accessed by browsing within the Program Editor as shown in section 3.4.3.
3.4.4 Setting up a plate layout The role of the Plate Layout Editor is to define what types of samples go in to which wells of the plate. It is represented in the top-right panel of the Experiment Editor. The plate layout can be cleared or changed before or after the run. As with the program setup, the plate layout can be defined in three ways: Click this button when the plate layout is to be defined entirely from new. Click Browse to search the plate layout library files for existing templates.
• Show all wells: The well information table will show only the details for the currently selected sample type, e.g. standards, unless this button is clicked. Click again to change back. • Reset names: Allows any user-defined changes to the names to be returned to the default. • Function buttons: Icons on the left-hand side provide easy access to basic functions (further details in section 3.4.4.6). Click once to activate. 3.4.4.
• Calibrator (CAL): In relative quantification methods, the ratio of the DNA template in different samples may be normalized to a calibrator sample. Particularly useful when comparing Cq values (section 4.7). • Reference Gene (REF): A common gene that is expressed in all samples at the same level. Can be useful in relative quantification methods. 3.4.4.
3.4.4.5 Replicates Choose how many replicates there are for each sample from the scroll-down menu. The Next group added to the plate layout will run in numerical order unless otherwise specified. 3.4.4.6 Assigning sample types to wells • Click on a sample type icon, for instance, the UNK icon, and the icon will become highlighted to show it is active. • Click on or drag over the wells of the plate with the mouse and the corresponding colour and number will appear in each well.
Undo: Cancels the previous operation. Rotate plate 180°: Useful if the plate was loaded into the instrument the opposite way round to the designated layout. These functions can also be accessed from the Tools option in the menu bar. 3.4.4.7 Well information table As a sample is allocated to each well, information about the sample will appear in the adjacent table. A default name and number is assigned e.g. Standard 1, but this can easily be changed by highlighting and typing over the text.
3.4.4.8 ® Importing/exporting plate layouts from/to Microsoft Excel Sample information can be copied into the Plate Layout Editor from an Excel spread sheet. The Excel file must be in the following format to permit correct import of sample types and well information: • To import from Excel into the Plate layout, select the cells to be copied and press Ctrl+C or select Copy.
3.4.4.9 Browse for an existing plate layout The user can browse for an existing plate layout file by clicking on the Browse button in the Plate layout pane of the Experiment Editor. This will open up the Plate Layout library folder and display any existing templates (.qpla files). • Highlight a file and click Open to open up the plate layout file in Experiment Editor. The template is ready for use. 3.4.4.
IMPORTANT: Certain characters must not be used in the file name otherwise it may become corrupted and you may not be able to open the Results file. These characters are: < > & ‘ “ • Change the file name as required. If an existing name is selected then a warning message will appear asking if the file should be over-written. The file (.qpla format) will automatically be saved to the directory My Documents\Quansoft\Plate Layouts.
3.4.5 Defining the analysis method 3.4.5.1 Selecting an analysis method The Analysis Selection box in the Experiment Editor (also found in the Results Editor after the run has finished) allows the user to define the method of analysis to be applied to readings gathered during the PCR program. • Analysis Selection box: Displays the stage name as assigned in the program setup and any analysis method setup.
• Analysis Method: The drop-down menu allows an analysis method to be selected. • Dye name: The name of the dyes selected in the program setup will be displayed. • Dye Usage: Assign a Dye Usage from the list in the drop-down menu. There will be one dye usage box for each read present in the selection stage. • Cancel: Aborts the procedure and takes the user back to the Experiment Editor. • Finish: Accepts all the default analysis settings for the analysis method chosen and closes the Wizard.
• Plus/minus scoring • Allelic discrimination • Multi-read See Chapter 4 for a specific discussion of each analysis method in terms of requirements, setup and what each approach can reveal about the experimental data. 3.4.5.3 Assigning a dye usage As there may be more than one dye reading in a single stage, the role of each dye needs to be assigned. To do this, the Analysis Wizard Selection box has a drop-down menu containing a list of definitions for each.
2. Passive reference dye (PRD) correction: • Check the box if this correction method is required (only available if a PRD was selected in the dye usage table). The purpose of the PRD is to normalize the reporter fluorescence and make well-to-well comparisons more accurate. The readings are normalized by dividing the fluorescence of the reporter in each well by that of the PRD. This correction method is applicable to all the analysis types available on PrimeQ (see section 4.4 for more details). • Click Next.
Repeat the process for additional stages. 3.4.6 Saving an experiment to the library The experiment is now complete with a program file, plate layout and analysis method all defined. While the program and plate layout can be saved as separate files (shown in 3.4.3.10 and 3.4.4.12), the experiment can also be saved as a consolidated .qexp file at any point during the setup in the Experiment Editor.
• Click Edit in the program or plate layout pane of the Experiment Editor and the appropriate editor will open. • Change settings in any step/stage, alter readings and so forth or delete the current template and import another from the library files. • Analysis: Either double-click the name or highlight and press Edit. Make any changes in the same way as when setting up the analysis. Change the title of experiment if you do not want to over-write the original file and add any comments if required.
3.5 Running an experiment 3.5.1 Starting the run 3.5.1.1 Preparing the system • Turn on the power to PrimeQ using the power switch at the rear of the instrument. An idle screen will appear on the instrument’s LCD display. • Turn on the PC and log in under a user name. • Double-click the Quansoft icon located on the PC desktop to open the software. • If the instrument has not been used previously, the filter cartridges will need to be installed (section 1.16).
3.5.1.3 Running an experiment 3.5.1.4 Run to report Unless otherwise informed, the system will wait for an acknowledgment from the user that the run has ended before proceeding on to analysis and report generation. If Quansoft is required to run straight through to report generation with no prompt or user intervention then select the Run to Report option found under Tools on the Experiment Editor menu bar.
• Save to the Experiment library folder if required (see section 3.4.6). Note that saving will over-write the previous version of the experiment. IMPORTANT: Certain characters must not be used in the file name otherwise it may become corrupted and you may not be able to open the Results file. These characters are: < > & ‘ “ A box will now appear asking to save the results in the Results library. • Change the file name of the Results file if required.
Experiment Editor and assign cartridges to each read. Alternately when the run button is pressed the following error window will appear: Selecting OK will take the user to the correct window where all of the reads can be assigned a filter cartridge. 3.5.2 Monitoring the run 3.5.2.1 From the instrument Progress and current status is reported via the display screen on the front of the instrument. • Cycle cc/mm: Reflects the current cycle number out of the total number for that stage eg.
3.5.2.2 From the Run Screen As the PCR is in progress, the Run Screen displays the current status of the program in real time. Fluorescence data is shown on a per-well basis with the different stages of the run (which have readings) being displayed as separate tabs. The user can select an individual well to show the fluorescence data for that well only. The readings are time-stamped and saved in the results data.
• Fluorescence graph: Data for a particular stage will be displayed in the graph. Choose to display readings for the entire plate or just for selected wells. Change the dye being viewed using the Dye to View option. • Well view: A fluorescence curve is displayed in each well as the PCR progresses. Highlight a well(s) with the mouse to display the fluorescence data for that well(s) in the adjacent graph. To return to viewing all filled wells, click just outside the top left-hand corner of the plate view.
3.6 LED intensity settings Please note the following: • If the fluorescent counts on the raw data plot of the Run Screen or Results Editor are above 100,000 counts using the Medium LED setting, it is recommended that the Low setting be used for the assay instead. • Similarly, if there are less than 1,000 counts on the Medium setting, use the High LED setting instead.
3.7 Results Editor 3.7.1 Post-run analysis main screen At the end of a run, if an analysis method was assigned to a stage with a read, the analysed readings (calculated using either the software defaults or parameters set by the user prior to the run) will be presented in the Results Editor. If no analysis method was setup, only the raw fluorescence data will be displayed.
A. User and experiment ID: A user name can be entered and comments specific to the experiment can be added in the Comments box. B. Results tabs: To the left of the main screen tab are a series of Results Editor tabs. The uppermost (active) tab displays the Result Editor page as shown. The tabs then list the stages in the order that they ran. Clicking on the tabs will display the results and graphs relevant to that particular stage. It is also possible to move between stages from the Results Editor menu bar.
results table as a strike-through). This is an important tool for selectively eliminating a well from the calculations. F. Results table: Contains the calculated results for the run. • The results table can be maximized for improved visibility. • Use the Combine Replicates function to display an average of replicate readings (click again to revert). • The calculated results will vary according to the method of analysis – see Chapter 4 for analysis-specific explanation. G.
name. This function can be turned off by clicking on the padlock icon. The user can also choose to save the experiment as just an experiment file i.e. minus the results. On the File option on the menu bar, choose Save as Experiment File and the data will be saved as a .qexp file into the default Experiment Library folder. If the padlock icon is locked then the file cannot be given the same name as an existing file in the destination folder.
• If more sophisticated editing functions are required, access the Graph Properties… option from the Results Editor menu bar. Select Analysis and then Graph Properties… The graph editing box is equipped with many features that allow the user to edit different aspects of the graph including its appearance, the data itself or the print and export options. One of the most important features is the Export tab which enables the user to export the graphs in different file formats. 3.7.
Clicking the PAR button brings up the analysis parameters relevant to the analysis being performed. The parameters can be edited with changes reflected immediately in the adjacent graph(s). 3.7.4.2 Change the analysis method An entirely different analysis method can be chosen or existing parameters edited from the Analysis Selection box found on the Results Editor main page.
3.7.5 Log/Audit trail Clicking on this tab displays all the information about the run that may be required for GLP purposes. It is separated into an Experiment Log, Instrument Log and Audit Trail. • Experiment Log: User and experiment details. • Instrument Log: Details of the program such as stage name, number of cycles, temperature and ramp rate. • Audit: Includes a summary of the experiment details such as the user, date, time, old/new parameters which have been selected.
o Summary of filters used at each stage, emission and excitation wavelengths and user-defined name o Integration time • Table of results 3.7.6.1 Report layout 3.7.6.2 Report screen function buttons Frequently used functions are found on the Task bar: Show current page number out of total. Go to first page/previous page. Go to next page/go to last page. Export to .pdf. The report options can easily be changed from within the report tab.
The Report Options box: change how the data is displayed using these settings. 3.7.6.3 Run to report If this option has been chosen on setup (from the Tools option on the Experiment Editor menu bar), at the end of the run, the software will automatically analyse the data and run through to report based on the user-defined options during setup. If no analysis has been set then only the plate layout and fluorescence data will be shown. 3.
o Click on Start/All Programs/Techne/Quansoft/Utilities/Data Extractor. o Browse for the Results File name and click Open. Individual tabs show details of the experiment, plate layout and stages with readings. o Use the drop-down menu to select the readings from different dyes. o Click on Copy to copy the data to the clipboard. The data can then be pasted into Excel. 3.8.
Data analysis Data analysis About this chapter This chapter looks in more detail at the different analysis methods available to users of PrimeQ. Following a brief introduction providing a reminder of the theory behind real-time PCR data collection, the chapter goes on to discuss each analysis in more detail including setup, applications and tips for hands-on use. Relative Fluorescence (Log) 10 1 0.1 0.01 0.001 0.
3.9 Introduction PrimeQ can be used to determine the absolute or relative quantity of a target DNA template in a given test sample by measuring the cycle-to-cycle change in the fluorescent signal. It can also measure the simple presence or absence of a target.
The crossing line is a best-fit of where the reaction efficiency is at its highest and most constant for each reaction curve. The quantification cycle, or Cq, (sometimes referred to as the CT in the literature), is the point at which an amplification curve intercepts the crossing line. Measuring fluorescence at the Cq for each well will allow the most reliable well-to-well comparison.
the early stages is expanded and the Cq value can be determined more accurately. Although some values may be negative when plotted on a log scale (especially if the data has been background corrected) this will not affect the accuracy of the data. 3.9.4 First derivative maximum Simple to perform and requiring no user input, this method determines the quantification cycle by calculating the point on the reaction curve at which the rate of change in fluorescence is fastest.
of the GC content, length and sequence of a DNA product, it is a useful tool in product identification. Dissociation curve showing the dissociation peaks. 2,500 -dF/dT 2,000 1,500 1,000 500 0 82 84 86 88 Tem perature 90 92 At the start of an analysis, the reaction is at a low temperature such that the DNA in the reaction tube will be double-stranded and the fluorescence signal high.
3.10 Choosing an analysis method Quansoft allows the user to assign an analysis method to any stage of the PCR that has fluorescent readings. The available analysis methods are: • None: When no analysis method has been set, the Results Editor will display the plate layout and a graph of raw fluorescent data. • Baseline correction: Allows the user to adjust the data for background fluorescence.
3.10.1.1 General points • Analysis methods can be added or changed post-run as long as the assay setup is valid for the method (see above table). • If multiple dyes are to be corrected differently within a stage then this must be performed post-run. • Data can be re-analysed an unlimited number of times – simply ensure any graphs or calculations of interest have been saved and change any analysis parameters or methods via the Analysis Selection box in the Results Editor.
3.11 Analysis method: None The default analysis method is None, so analysis parameters do not need to be set. 3.11.1 Viewing the results During the run, the real-time collection of data can be monitored in the Run Screen. The plate layout shows the fluorescence curve on a per-well basis and the temperature profile plot indicates how far the run has progressed. When the run has completed, results can be viewed in the Results Editor with data from each stage of the run located under its own tab.
• Clicking the block temperature function button brings up the block temperature graph in the lower pane. 3.11.2 PrimeQ Report The PrimeQ Report shows the plate layout and raw fluorescence data with the default run details displayed by scrolling down the screen. See section 3.7.6.
3.12 Passive reference dye (PRD) correction The purpose of the passive reference dye (PRD) is to normalize the reporter fluorescence so that well-to-well comparisons are more accurate. The readings are normalized by dividing the fluorescence of the reporter in each well by the PRD for each well. This correction method is applicable to all the analysis types available on PrimeQ except “None”.
3.13 Analysis method: Baseline correction This method allows the user to adjust the data for any background fluorescence. This approach looks at the signal in the early cycles of the PCR and then averages out the early noise and subtracts it from subsequent readings. As with passive reference dye (PRD) correction detailed above, baseline correction can be useful for correcting the data prior to performing an analysis and so can help to increase the accuracy of the assay. 3.13.
3.13.2.2 Baseline correction • Click Next. Options appear for baseline correction. • None: No correction. • Arithmetic 1: Subtracts the average of the specified range of readings from each well. • Arithmetic 2: Subtracts the average of a specified number of lowest readings from each well. • Proportional: The selected number of lowest readings is averaged and used to create a baseline while the selected number of highest readings is averaged to create a maximum.
3.13.2.3 Report Options and Summary • Clicking Next leads through to the Report Options screen. Baseline corrected data can be displayed in a 96-well or a graphical format – click the appropriate box to select. • Click Next to view a summary of the setup. • Click Back to change any settings or Cancel to abort the procedure. • Click Finish to complete the setup. The analysis method appears in the updated Analysis Selection box on the Experiment Editor main page (or Results Editor if post-run).
3.13.3.1 Viewing and changing the parameters • Click the PAR button next to the baseline correction graph to bring up the parameter settings for the analysis. If any settings are changed, the data will be recalculated and the graphs and results table updated accordingly. The settings or analysis method can also be changed by accessing the Analysis Selection box from the Results Editor main page.
3.13.4 PrimeQ Report 3.13.4.1 Display options The report options can be changed from within the report tab of the Results Editor. • Click on the report options icon, which will bring up the Report Options box. Tabs will display the report options relevant for each stage. Change as appropriate and click Done to finish. 3.13.5 Quick guide to baseline correction analysis 1.
3.14 Analysis method: Quantification This analysis method has two general approaches: either to determine the ‘absolute’ concentration of an unknown sample by comparison of the quantification cycle (Cq) to a standard curve of known concentrations or else a relative value determined by the comparison of Cqs.
3.14.2.1 PRD If a PRD was assigned in the dye usage box, the next screen displays an option for passive reference dye correction (see section 4.4). Clicking Next leads through to the Baseline Correction screen. 3.14.2.2 Baseline correction See section 4.5. Choose the method as required and set the values as appropriate. 3.14.2.3 Cq calculation The crossing line is a best-fit of where the reaction efficiency is at its highest and most constant for each reaction curve.
Setting the noise threshold and crossing line Overview of procedure: The noise threshold and crossing line are placed on the amplification graph either at positions specified by the user or using the default settings.
Calculating the quantification cycle Fit points are a defined number of fluorescent points found on the curve just above the crossing line. The points are used in a linear regression calculation and the interception of the linear regression through the fit points and crossing cursor provides a value for the quantification cycle. This will be a fractional number calculated for each well. All the user has to do is set the number of fit points to be included in the calculation and the software does the rest.
3.14.3 Viewing the results During the run, the real-time collection of data can be monitored in the Run Screen. The plate layout shows the fluorescence curve on a per-well basis and the temperature profile plot indicates how far the run has progressed. When the run has completed, results can be viewed in the Results Editor with data from each stage of the run located under its own tab. 3.14.3.
Changes to the analysis parameters can also be made via the Analysis Selection box on the main page of the Results Editor. If a PRD was assigned in the dye usage box, then the PRD correction options can be accessed by clicking the PAR icon next to the raw data graph. • Click the raw data graph icon if the raw data graph is not displayed. 3.14.4 PrimeQ Report The report options can be changed from within the report tab of the Results Editor.
Overview of procedure: The experiment is set up using the Quantification Wizard as detailed above. The experiment must include a dilution series of standards in the plate layout by which to compare the unknowns. The concentration of each standard should be added to the Well Information table in the Plate Layout Editor (can be accessed through the Results Editor main screen, post-run).
3.14.6.2 The standard curve y = -3.379x + 36.724 R² = 0.996 E = 1.977 26 Cycle Number 24 22 20 18 16 14 3 4 5 Log Concentration 6 7 Linear regression is used to generate a straight line, y = mx + c whereby: • y = the slope, ideally this should be approximately -3.32. • c = the value at which the line intercepts the y axis. 2 • R = Correlation coefficient (mean squared error of the determination) – a perfect correlation has a value of 1. -1/slope • E = efficiency of the reaction whereby E = 10 .
Cycle Number y = -3.402x + 43.278 R² = 0.999 E = 1.967 36 34 32 30 28 26 24 22 20 18 16 14 2 3 4 5 6 Log Concentration 7 8 9 3.14.6.4 Results table Concentrations are calculated for the unknowns based on a comparison of Cqs with standards of known concentration. The results are displayed in the results table. Column headings: • No: Well number. • Well ID: Location of well. • Sample ID: User-supplied or default name of well.
3.14.7 Comparing Cqs in relative quantification Cqs are also used for relative quantification i.e. the comparison of two different reporters in the same well. Although this approach can be performed either with or without a standard curve, it is particularly useful for screening assays where it is necessary to compare a fold difference of sample B to a calibrator sample A, for example.
• Click through to the Summary window and click Finish. 3.14.7.2 Viewing relative quantification results During the run, the real-time collection of data can be monitored in the Run Screen. The plate layout shows the fluorescence curve on a per-well basis and the temperature profile plot indicates how far the run has progressed. When the run has completed, results can be viewed in the Results Editor with data from each stage of the run located under its own tab.
The results table displays the calculated results. Column headings: • No: Well number. • Well ID: Location of well. • Sample ID: User-supplied or default name of well. • Cq (Dye 1): Quantification cycle for Dye 1. • Conc (Dye 1): Calculated concentration of sample for Dye 1. • Cq (Dye 2): Quantification cycle for Dye 2. • Conc (Dye 2): Calculated concentration of sample for Dye 2. • Ratio: Dye 1 divided by Dye 2. • Comments: User inputted text. 3.14.7.
Choosing Relative Cq in the Relative Quantification window brings up the settings box. The user can select which reporters to compare using the drop-down box shown above. The first is usually the gene of interest and the second the REF gene amplified from the same sample. The Cq of the second reporter selected will be subtracted from that of the first and the resulting ∆Cq value can then be compared to that of a calibrator (if a reference sample has been defined as a calibrator, CAL, in the plate layout).
• Dye to view options offer relative quantification cycles or individual reporter results and graphs. • Changing the Dye to View from Relative Cq to a single reporter displays the baseline corrected graph (if chosen in the setup) in addition to the quantification cycles graph for the individual dyes. Column headings: • No: Well number. • Well ID: Location of well. • Sample ID: User-supplied or default name of well. • Cq (Dye 1): Quantification cycle for Dye 1. • Cq (Dye 2): Quantification cycle for Dye 2.
3.14.8 Quick guide to quantification analysis 3.14.8.1 Quantification 1. In the Experiment or Results Editor Analysis Selection box, highlight the stage on which quantification analysis is to be performed and click Edit. 2. In the Analysis Wizard Selection box, choose Quantification from the drop-down menu and assign a use next to the appropriate dye name. Click Next. 3. Choose whether to correct the results with a PRD if a PRD was assigned in the dye usage box. Click Next. 4.
3.15 Analysis method: Dissociation curve Dissociation curve analysis can add to the information obtained from the PCR. Also known as melting curve analysis, it measures the temperature at which the DNA strands dissociate (i.e. the melting temperature or Tm). Using an intercalating dye and increasing the temperature in small increments, the PCR products can be seen to ‘unzip’ at a specific temperature.
3.15.1 Assay requirements • Need at least one reporter dye. • Need a stage in the program with a ramp read (with at least two readings). 3.15.2 Raw data for reporter Setup Background correction and optional curve smoothing filter Dissociation curve Dissociation peaks Tm Peak areas Report • Program a ramp read into the PCR program as described in section 3.4.3.6. • In the Analysis Selection box in the Experiment Editor, click on the name of the ramp stage and then press Edit.
Digital filter This is an option to smooth the raw data before it is presented. It is carried out using a SavitskyGolay curve smoothing algorithm, which fits a third order polynomial to a number of points either side of the data to be smoothed. This is similar to averaging points either side of a centre point but because a third order polynomial curve is used, the profile of the curve is retained. The user can set the number of points on which to smooth the data in the drop-down box.
• A1: Start temperature +10% ramp • A2: Start temperature +30% ramp For example, if the temperature ramps for 30°C between 50°C and 80°C, 10% would equate to 3°C and A1 would be positioned at 50 + 3 (53°C); 30% equates to 9°C and so A2 would be positioned at 50 + 9 (59°C) and so on. These cursors should be positioned as far apart as possible without including any of the dissociation data. B1–B2: These cursors determine the remaining fluorescence background after the DNA has dissociated.
3.15.2.4 Manual peak detect To use manual peak detect, select this option on the Dissociation Wizard Peak Detector screen. • Number of peaks to find: Up to a maximum of four. • Bin threshold: The user can specify a temperature range (°C) in which a peak will be considered valid. • Threshold filter: The user sets a threshold value to limit the size of the peaks detected (see below). The default is 5%.
Peak Headings Each peak cursor can be individually named by typing a name in the appropriate Peak Headings box. • Click on the PAR button to view the Peak Headings box. • Type in a name for the peak. The new name will then appear in the Results Table column heading and in the Report. This may facilitate identification of peaks corresponding to different PCR products.
Bin threshold The user can specify a temperature range (ºC) either side of a cursor in which a peak will be considered valid. For example, if the bin threshold is set to 80°C with a range of 0.5°C, then all peaks with a Tm between 79.5 and 80.5°C will be considered in the results. The default is 0.5°C. Peaks within the valid +/- temperature range are sent to the peak column results table.
• Choose the Peak Area option by checking the box. 3.15.2.6 Report Options and Summary • Clicking Next leads through to the Report Options screen, which allows the user to decide how the data appears in the PrimeQ report. • Click Next to view a Summary of the setup. • Click Back to change any settings or Cancel to abort the procedure. • Click Finish to complete the set up. 3.15.3 Viewing the results During the run, the real-time collection of data can be monitored on the Run screen.
3.15.3.1 Viewing and changing the parameters Click the PAR button next to the graphs to bring up the settings box for dissociation curve analysis. If any settings are changed, the data will be recalculated and the graphs and results table updated immediately. The settings or analysis method can also be changed by accessing the Analysis Selection box from the Results Editor main page. 3.15.4 PrimeQ Report The report options can be changed from within the report tab of the Results Editor.
3.15.5 Quick guide to dissociation curve analysis • In the Experiment or Results Editor Analysis Selection box, highlight the stage on which Dissociation Curve is to be performed and click Edit. • In the Analysis Wizard Selection box, choose Dissociation curve from the drop-down menu and assign a use next to the appropriate dye name. Click Next. • Background correction screen: Choose whether to smooth the data with a digital filter and define where to position the cursors for correction. Click Next.
3.16 Analysis method: Plus-minus scoring This type of assay is used to record the presence or absence of a PCR product. Input data can either be kinetic (whereby the number of readings is >1) or end-point (a reading taken at the end of the run). The assay uses fluorophore chemistry in the same way as in the other assays such that the fluorescence produced during the reaction will be proportional to the number of target DNA molecules.
3.16.2.1 Baseline correction The choice of baseline correction method is similar to that shown in the Baseline Correction Wizard (section 4.5) with the exception that Proportional is excluded. Click Next when the baseline correction is set. 3.16.2.2 Plus-minus analysis method This screen allows the user to choose which readings are to be used for results scoring. • End-point (default): Uses the last reading only (we recommend that >1 reading is used for accuracy).
3.16.2.4 Report Options and Summary • Click Next to lead through to the Report Options screen. The default is for the plus/minus table to be displayed but an option for a scaled data graph displaying the confidence thresholds is also available. • Click Next to view a Summary of the setup. • Click Back to change any settings or Cancel to abort the procedure. • Click Finish to complete the set up. 3.16.
Clicking an individual well or a selection of wells in the plate layout will highlight just the selected well(s) on the plus-minus graph. Clicking the Show all wells button will re-select all wells. The plus-minus graph 100 90 % Relative To NTC 80 70 60 50 40 30 20 10 0 Well If the plus-minus graph is not displayed, click on the icon to bring up the data: The plus-minus graph displays the values as points and the confidence threshold as sliding cursors (blue is the lower threshold and red, the upper).
3.16.3.1 Viewing and changing the parameters Click the PAR button next to one of the graphs to bring up the analysis settings for plus-minus scoring. If any settings are changed, the data will be recalculated and the graphs and results table updated accordingly. The settings or analysis method can also be changed by accessing the Analysis Selection box from the Results Editor main page.
3.16.5 Quick guide to plus-minus scoring analysis 1. In the Analysis Selection box, highlight the stage on which plus-minus scoring is to be performed and click Edit. 2. In the Analysis Wizard Selection box, select Plus-minus scoring from the drop-down menu and assign a use next to the appropriate dye(s) name. Click Next. 3. If a PRD was assigned, the PRD correction screen will be displayed. Choose whether to perform a PRD correction on the data and click Next. 4.
3.17 Analysis method: Allelic discrimination Allelic discrimination is a technique that is able to detect single base pair differences. It is used to discriminate between genotypes, mutations and polymorphisms within or between samples. Conventional approaches to genotyping and mutation have traditionally relied on a three-stage process: amplification (e.g. PCR), discrimination (e.g. restriction digest) and detection (often radioactive), which are often time-consuming, complicated and costly.
• Click Next and the Allelic Discrimination Wizard will launch. 3.17.2.1 Baseline correction The baseline correction methods are the same as for plus-minus scoring. • Choose the appropriate method and click Next. 3.17.2.2 Allele scoring method The purpose of this part of the analysis setup is to define which readings should be used for allele scoring. The options displayed are the same methods used as in plus-minus scoring.
3.17.2.4 Report Options and Summary • Click Next to lead through to the Report Options screen. The default is for an allelic discrimination graph and a table of results. • Click Next to view a Summary of the setup. • Click Back to change any settings or Cancel to abort the procedure. • Click Finish to complete the set up. 3.17.3 Viewing the results During the run, the real-time collection of data can be monitored on the Run screen.
3.17.3.1 Allelic discrimination scatter graph Particular to the allelic discrimination method is the display of a scatter plot representing the fluorescence of one allele plotted against the other. The software plots the results of the run on a scatter plot of allele 1 vs. allele 2 (axes defined by the user in Reporter Selection) with each well of the 96-well plate represented by a point on the graph.
copies of both alleles and therefore produce fluorescence with both dyes. Samples that do not cluster tightly may contain rare sequence variations, sequence duplications or the PCR may not have been optimal. Assigning Genotypes The user can define clusters of points within the plot that represent genotypic segregation within the samples; the scoring of each sample is then presented in the Results table. • Click Type Colour in the Display box. • Select one of the coloured Type tiles in the Type box.
Results table For each well a fluorescence value for each reporter dye is displayed in the Results table together with its allele type determined from the typing assigned in the scatter graph. To change the order of the axes, enter the analysis setup using the PAR button or start the Allelic Discrimination Wizard from the Analysis Selection box on the Results Editor main screen. In Reporter selection change the order of alleles to compare. 3.17.3.
3.17.5 Quick guide to allelic discrimination analysis 1. In the Experiment or Results Editor Analysis Selection box, highlight the stage on which allelic discrimination is to be performed and click Edit. 2. In the Analysis Wizard Selection box, choose Allelic Discrimination from the drop-down menu and assign a use next to the appropriate dye(s) name. Click Next. 3. Baseline correction: Choose which method to use (proportional is not an option in allelic discrimination) and click Next. 4.
3.18 Analysis method: Multi-read This analysis method averages all or a selected range of values in the stage for a given well. Multiread is a useful tool in end-point analysis; end-point fluorescence values can be compared to Cq values obtained from quantification analysis of the amplification stage and used to aid primer optimization experiments. Alternatively, Multi-Read allows PrimeQ to be used simply as a plate reader. 3.18.1 Assay requirements • Need at least one reporter.
• End-point: Uses the last reading only (we recommend that >1 reading is used for accuracy). • All readings (default): Averages all readings in the stage • Last readings: Averages a user-specified number of last readings (the default is 5, or 1 if there are less than 5 readings) • Specify range: Specify a range of readings to be averaged that best suit the data (the default is the last reading). 3.18.2.2 Report Options and Summary • Click Next to lead through to the Report Options screen.
When the run has completed, results can be viewed in the Results Editor with data from each stage of the run located under its own tab. Multi-read graph The graph displays the average readings for each well as colour-coded points. If the multi-read graph is not displayed, simply click on the Multi-read icon to bring up the data: • Clicking a well(s) in the plate layout will highlight the selected well on the multi-read graph.
3.18.3.1 Viewing and changing the parameters Click the PAR button next to the Multi-Read graph to bring up the analysis settings. If any of the settings are changed, the data will be recalculated and the graphs and results table updated accordingly. The settings or analysis method can also be changed by accessing the Analysis Selection box from the Results Editor main page.
3.18.5 Quick guide to multi-read analysis 1. In the Experiment or Results Editor Analysis Selection box, highlight the stage on which multiread analysis is to be performed and click Edit. 2. In the Analysis Wizard Selection box, choose Multi-Read from the drop-down menu and assign a use next to the appropriate dye(s) name. Click Next. 3. Passive reference dye correction: If a PRD was assigned in the dye usage screen, options for correction will be displayed. Click Next. 4.
3.19 Multiplex assays Multiplex assays (or multi-colour detection) combine the use of two or more different reporters in the same well. PrimeQ’s cartridge carousel can house up to four pairs of excitation and emission filters, and as such, is capable of detecting up to four different reporters. Multiplexing is suitable for all analysis methods as long as different reporters are used with different emission spectra and that if more than one PCR is occurring in the same well, the reactions do not compete.
3.19.1.1 Viewing the results • Dye to View: Use this option to view the data for the individual reporters. • Results table: Results for only the dye currently being viewed will be listed in the results table unless an analysis method has been selected where more than one reporter is included in the calculation e.g. relative quantification, relative quantification cycles, allelic discrimination etc. 3.19.1.
4 Technical information Technical information on PrimeQ About this chapter This chapter provides all the technical information you may need to know about your PrimeQ real-time PCR system. It covers everything from spare parts to recommended consumables and maintenance routines.
4.1 Operator maintenance NOTE THAT THIS EQUIPMENT SHOULD ONLY BE DISMANTLED BY PROPERLY TRAINED PERSONNEL. REMOVING THE CASE EXPOSES POTENTIALLY LETHAL MAINS VOLTAGES. THERE ARE EQUIPMENT. NO OPERATOR MAINTAINABLE PARTS WITHIN THE In the unlikely event that you experience any problems with your unit which cannot easily be remedied, you should contact your supplier and return the unit as necessary.
4.1.3 Insulation Testing This equipment is fitted with RFI suppression circuitry. Any check of the electrical insulation by means of high voltage dielectric testing must be carried out using only a DC voltage. This unit contains semiconductor components which may be damaged by electric field effects. 4.1.4 Mantenimiento ESTE APARATO DEBE SER DESMONTADO SOLO Y EXCLUSIVAMENTE POR PERSONAL DEBIDAMENTE CAPACITADO.
2. Fusibles La protection de l’appareil est assurée par deux fusibles dont le remplacement ne peut être effectué que par un personnel qualifié. Si les fusibles sautent sans arrêt, il s’agit d’un problème sérieux. Nous vous conseillons dans ce cas de prendre contact avec votre fournisseur pour réparation. 4.2 User responsibilities • To use the instrument in such a manner as to minimize interference with the optical system.
4.4 Minimum computer requirements A PC is not supplied with PrimeQ The following are the recommended minimum PC specifications required for running PrimeQ: CPU: Core 2 Duo or equivalent Memory: 2Gb DDR RAM as a minimum Storage: 40Gb DMA hard drive Display: 1024 x 768 resolution minimum, 17 inch digital monitor recommended.
4.
4.7 Packing the PrimeQ instrument 4.7.1 Remove the filter cartridges Before packing the PrimeQ for transit it is essential to remove the filter cartridges. • In the Cartridge Access and Editing screen of the Filter Wizard, click Remove next to the appropriate filter. Follow the procedure as shown and click Finish to complete. A message will appear informing the user to ‘Please wait – checking cartridge status’.
4.7.2 Packing the instrument in the carton Before placing in the carton, ensure that the drawer is fully closed. This prevents movement which could cause damage to the drawer locking mechanism • Pack the filters; close the drawer: Place the filters in the storage box. • Bag the PrimeQ and place into the carton base: The carton has a shaped base. Wrap the PrimeQ in the anti-static bag and place in the base of the carton. Place a piece of foam in front of the drawer to prevent it from opening.
Check-list Have you: 1. Removed the filters? 2. Closed the drawer? 3. Used the anti-static bag? 4. Packed the unit correctly? 5. Packed all the accessories including the filter cartridges? 6. Strapped to a pallet? 4.8 Packaging On all correspondence, please quote the serial number in full and/or the Sales Order Number.
5 Troubleshooting Troubleshooting real-time PCR and PrimeQ About this chapter This chapter contains information required for troubleshooting the real-time PCR process, the control software and the instrument itself.
5.1 Troubleshooting ISSUE CAUSE SOLUTION No display on LCD screen. No power to instrument. Check power supply is connected. Fuse blown in plug. Check fuse and change if appropriate. Incorrect reaction conditions. Optimize assay and run agarose gel to check PCR Reaction component not added. Check correct reagents were added. Incorrect primer or probe sequence. Redesign primers or probe. No template added to reaction or degraded template present. Repeat assay.
ISSUE CAUSE SOLUTION No Cq recorded for a sample. No template added. Repeat assay. No target in sample. Noise and crossing line set incorrectly. The default settings in Quansoft are 4 and 10 standard deviations respectively, adjust these through the parameter option or the Analysis Wizard. Decreased volume in samples at end of a run. Poor seal. Ensure the sealing method is appropriate. Heat sealing is recommended. Higher Cq than expected. Fewer templates added to reaction.
ISSUE CAUSE SOLUTION Instrument cannot be seen in the prerun screen or in the Cartridge Access and Editing screen. Connection lost between instrument and computer. Restart instrument and computer. Fatal error displayed on LCD screen. Instrument error. Note the error message and contact your Techne Distributor. Any other error which prevents a PCR run being initiated. Instrument error. Open Windows Explorer.
5.2 Real-time PCR Glossary Absolute quantification A standard curve is used to quantify unknown samples by interpolating their quantity. Allelic Discrimination Technique used in genotyping to distinguish the presence or absence of two alleles using two different dye-labelled probes. Amplicon Segment of DNA generated by PCR. Amplification plot Graphical representation of fluorescence signal versus cycle number.
PCR Efficiency An efficient PCR assay shows a doubling in the specific product after every cycle: -1/slope Reaction efficiency E = 10 ideal value is 2 The efficiency is affected by the length, GC content and secondary structure of the amplicon and sub-optimal reaction conditions. PRD 2 Passive Reference Dye: Internal reference dye to which the reporter dye is normalized to correct for changes in volume and block variations.
