Techne PrimeQ Manuale Utente

Version 5.0 06/14 PrimeQ OPERATOR’S MANUAL

104.3.2 PrimeQ Report ... 112 4.4 Passive refer

100o Summary of filters used at each stage, emission and excitation wavelengths and user-defined name o Integration time • Table of results 3.7.6

101 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 ch

102o Click on Start/All Programs/Techne/Quansoft/Utilities/Data Extractor. o Browse for the Results File name and click Open. Individual tabs show

103 Data analysis Data analysis About this chapter This chapter looks in more detail at the different analysis methods available to

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105 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 measur

106The crossing line is a best-fit of where the reaction efficiency is at its highest and most constant for each reaction curve. The quantification c

107 the early stages is expanded and the Cq value can be determined more accurately. Although some values may be negative when pl

108of the GC content, length and sequence of a DNA product, it is a useful tool in product identification. Dissociation curve sh

109 3.10 Choosing an analysis method Quansoft allows the user to assign an analysis method to any stage of the PCR that has fluoresce

114.10.5 Quick guide to multi-read analysis ... 161 4.11 Multiplex assays ...

1103.10.1.1 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)

111 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 Durin

112• Clicking the block temperature function button brings up the block temperature graph in the lower pane. 3.11.2 PrimeQ Report The PrimeQ Repo

113 3.12 Passive reference dye (PRD) correction The purpose of the passive reference dye (PRD) is to normalize the reporter fluorescence so that well

1143.13 Analysis method: Baseline correction This method allows the user to adjust the data for any background fluorescence. This approach look

115 3.13.2.2 Baseline correction • Click Next. Options appear for baseline correction. • None: No correction. • Arithmetic 1: Subtracts the ave

1163.13.2.3 Report Options and Summary • Clicking Next leads through to the Report Options screen. Baseline corrected data can be displayed in a 96

117 3.13.3.1 Viewing and changing the parameters • Click the PAR button next to the baseline correction graph to bring up the param

1183.13.4 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 t

119 3.14 Analysis method: Quantification This analysis method has two general approaches: either to determine the ‘absolute’ concentration of an unkn

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1203.14.2.1 PRD If a PRD was assigned in the dye usage box, the next screen displays an option for passive reference dye correction

121 Setting the noise threshold and crossing line Overview of procedure: The noise threshold and crossing line are placed on the amplific

122Calculating the quantification cycle Fit points are a defined number of fluorescent points found on the curve just above the crossing line. The

123 3.14.3 Viewing the results During the run, the real-time collection of data can be monitored in the Run Screen. The plate layo

124 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

125 Overview of procedure: The experiment is set up using the Quantification Wizard as detailed above. The experiment must include a dilution series

1263.14.6.2 The standard curve Linear regression is used to generate a straight line, y = mx + c whereby: • y = the slope, ideally this should be

127 3.14.6.4 Results table Concentrations are calculated for the unknowns based on a comparison of Cqs with standards of known concentration. The

1283.14.7 Comparing Cqs in relative quantification Cqs are also used for relative quantification i.e. the comparison of two different r

129 • Click through to the Summary window and click Finish. 3.14.7.2 Viewing relative quantification results During the run, the real-time co

131 Introduction Safety and installation information About this chapter This chapter provides information on general safety aspects, defi

130 The results table displays the calculated results. Column headings: • No: Well number. • Well ID: Location of well. • Sample ID: User-supplied

131 Choosing Relative Cq in the Relative Quantification window brings up the settings box. The user can select which reporters to c

132 • Dye to view options offer relative quantification cycles or individual reporter results and graphs. • Changing the Dye to Vie

133 3.14.8 Quick guide to quantification analysis 3.14.8.1 Quantification 1. In the Experiment or Results Editor Analysis Selection box,

1343.15 Analysis method: Dissociation curve Dissociation curve analysis can add to the information obtained from the PCR. Also known as

135 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.

136 Digital filter This is an option to smooth the raw data before it is presented. It is carried out using a Savitsky-Golay curve smoothing algorit

137 • A1: Start temperature +10% ramp • A2: Start temperature +30% ramp For example, if the temperature ramps for 30°C between 50°C and 80

138 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 t

139 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 v

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140Bin threshold The user can specify a temperature range (ºC) either side of a cursor in which a peak will be considered valid.

141 • 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 s

142 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

143 3.15.5 Quick guide to dissociation curve analysis • In the Experiment or Results Editor Analysis Selection box, highlight the stage

1443.16 Analysis method: Plus-minus scoring This type of assay is used to record the presence or absence of a PCR product. Input data can eithe

145 3.16.2.1 Baseline correction The choice of baseline correction method is similar to that shown in the Baseline Correction Wizard (

146 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

147 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. Clicki

148 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 pl

149 3.16.5 Quick guide to plus-minus scoring analysis 1. In the Analysis Selection box, highlight the stage on which plus-minus scoring

151.1 PrimeQ PrimeQ - the real-time nucleic acid detection system from Techne - has been designed with the advantage of an open chemistry form

1503.17 Analysis method: Allelic discrimination Allelic discrimination is a technique that is able to detect single base pair differences. It is use

151 • Click Next and the Allelic Discrimination Wizard will launch. 3.17.2.1 Baseline correction The baseline correction methods are the same as f

152 3.17.2.4 Report Options and Summary • Click Next to lead through to the Report Options screen. The default is for an allelic discr

153 3.17.3.1 Allelic discrimination scatter graph Particular to the allelic discrimination method is the display of a scatter plot re

154copies of both alleles and therefore produce fluorescence with both dyes. Samples that do not cluster tightly may contain rare sequ

155 Results table For each well a fluorescence value for each reporter dye is displayed in the Results table together with its allele type determined

1563.17.5 Quick guide to allelic discrimination analysis 1. In the Experiment or Results Editor Analysis Selection box, highlight the stage on whic

157 3.18 Analysis method: Multi-read This analysis method averages all or a selected range of values in the stage for a given well. Multi-read is a u

158 • End-point: Uses the last reading only (we recommend that >1 reading is used for accuracy). • All readings (default): Averages a

159 When the run has completed, results can be viewed in the Results Editor with data from each stage of the run located under its ow

161.2.3 Program Editor Individual cycle steps, stages or temperature ramps can be added quickly and easily together with other parameters to build a

160 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 an

161 3.18.5 Quick guide to multi-read analysis 1. In the Experiment or Results Editor Analysis Selection box, highlight the stage on which multi-rea

1623.19 Multiplex assays Multiplex assays (or multi-colour detection) combine the use of two or more different reporters in the same well. PrimeQ’s c

163 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 on

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165 4 Technical information Technical information on PrimeQ About this chapter This chapter provides all the technical information you may

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167 4.1 Operator maintenance NOTE THAT THIS EQUIPMENT SHOULD ONLY BE DISMANTLED BY PROPERLY TRAINED PERSONNEL. REMOVING THE CASE EXPOSES POT

1684.1.3 Insulation Testing This equipment is fitted with RFI suppression circuitry. Any check of the electrical insulation by means of h

169 2. Fusibles La protection de l’appareil est assurée par deux fusibles dont le remplacement ne peut être effectué que par un personn

171.3 Unpacking When unpacking the unit, check that the following have been removed from the packing: • This PrimeQ operator guide • A CD contai

1704.4 Minimum computer requirements A PC is not supplied with PrimeQ The following are the recommended minimum PC specifications required

171 4.6 Replacement parts The following replacement parts can be obtained from Techne or your Techne dealer: Part number Description Quantity req

1724.7 Packing the PrimeQ instrument 4.7.1 Remove the filter cartridges Before packing the PrimeQ for transit it is essential to remove the filter

173 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 co

174Check-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 t

175 5 Troubleshooting Troubleshooting real-time PCR and PrimeQ About this chapter This chapter contains information required for troub

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177 5.1 Troubleshooting ISSUE CAUSE SOLUTION No display on LCD screen. No power to instrument. Check power supply is connected. Fuse blown in

178ISSUE CAUSE SOLUTION No Cq recorded for a sample. No template added. Repeat assay. No target in sample. Noise and crossing line set incor

179 ISSUE CAUSE SOLUTION Instrument cannot be seen in the pre-run screen or in the Cartridge Access and Editing screen. Connection lost between ins

181.7 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

1805.2 Real-time PCR Glossary Absolute quantification A standard curve is used to quantify unknown samples by interpolating their quantity. Alle

181 PCR Efficiency An efficient PCR assay shows a doubling in the specific product after every cycle: Reaction efficiency E = 10-1/slope ideal value

19The 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 DANGE

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201.9.3 Operator safety All users of Techne equipment must have available the relevant literature needed to ensure their safety. It is important

21The fused plug supplied with the mains cable is fitted with a 10 amp fuse to protect the instrument and the operator. The rating p

22Le 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 10

231.10 Before switching on 1.10.1 PrimeQ front view 1.10.2 Fuses Ensure that the correct fuses are fitted for your voltage supply. Fus

241.11 Technical Specification Thermal cycler Block Format: 96 x 0.2ml well low-profile micro tube plate Block Specification: 8 x Peltier block emp

25Computer requirements (Not Supplied) The following are the recommended minimum PC specifications required for running PrimeQ: CPU: Core 2 Duo or e

26Within these periods we undertake to supply replacements free of charge for parts which may on examination prove to be defective, provided that the

271.14 Installing the application software Always set your PC or laptop from which you are running PrimeQ so that standby is switched OFF. This can e

28 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

29The unlock code can be obtained in one of two ways: 1. E-mail: [email protected] an email complete with details of your nam

3 How to use this guide Please read all the information in this guide before using the unit. Le rogamos lea cuidadosamente la información conte

301.15 Using the LCD control panel 1.15.1 Function keys PrimeQ is controlled primarily from the PC using the application software, althou

311.15.4 Before you start Check that: 1. All cables are connected between the instrument and PC. 2. All power cables are plugged in and comply wi

321.16.2 Filter cartridge care instructions • The filter cartridges must be handled with special care. • Only lift them from the foam in the box b

33• Excitation/emission wavelengths: As defined in the Add Cartridge settings box. • Dye name: Each cartridge can be assigned up to four d

341.16.5 Assigning filter descriptions in Quansoft Once the cartridge has been fitted into the cartridge carousel, the system will chec

35A message will appear informing the user to Please wait – checking cartridge status. If the removal was performed correctly, the Cartridge Access a

36 • 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 th

371.18 LED power settings The LED light source has variable power settings. This prevents PMT blinding and brings fluorescence into it

381.20 After use When the samples have finished thermal cycling, remember that parts of the unit, such as the tubes, blocks and associated accessorie

392 Getting started Introduction to PrimeQ and real-time PCR About this chapter This chapter gives a description of PrimeQ together wit

4Chapter 6 Troubleshooting and Glossary Identifying problems with the instrument and/or the PCR Glossary of terms used in real-time PCR

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412.1 Introduction to PrimeQ 2.1.1 Principle of a real-time PCR instrument A real-time polymerase chain reaction (PCR) system performs three main f

422.2 Introduction to Real-time PCR 2.2.1 PCR PCR is a powerful biochemical technique that has revolutionised biological research by allow

43PCR (qPCR) extends the usefulness of the technology by permitting the reliable determination of starting DNA template. 2.2.3 Real-time PCR on Prim

44dye is excited at the appropriate wavelength should be proportional to the quantity of target DNA present. For each type of fluorescent chemistry,

45fluorescence signal or fluorescence at a longer wavelength which is not detected. The 5’ fluorophore is often called the reporter. Mo

462.4 System overview PrimeQ is a real-time PCR unit into which the operator places a 96-well plate containing the samples to be anal

472.5.1 The optical light path Excitation light from the solid state white light source (1) passes through a light path and enters the

48PrimeQ’s thermal cycler block accommodates a low-profile 96-well plate. Each block is internally calibrated so that if the block is replace

493 Quansoft Using Quansoft About this chapter Accompanying PrimeQ is the intuitive, wizard-based software, Quansoft. This chapter discu

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513.1 Introducing Quansoft PrimeQ is a real-time-PCR unit containing sophisticated thermal cycling and fluorescence detection technology.

52 Plate Layout Editor Within a matter of seconds, a 96-well PCR plate can be assigned with various sample types, including blanks, c

533.2.1 Home page As shown in the schematic diagram above, the Quansoft Home page is the starting point for an experiment, providing quick li

54 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

55Quansoft’s Results Editor displays the results of the run, which can be sent to report or re-analysed with different parameters.

563.3.2 Main screen Shortcuts for experiment setup and data analysis: Run an experiment: goes to the Experiment folder to select an experiment. C

57 Clicking the Security icon allows customizing of the supervisor password (required for the administrative functions which are acces

58 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 c

59 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 d

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603.4 Setting up an experiment 3.4.1 An Overview Schematic diagram showing the options available for setting up an experiment on PrimeQ. The ic

613.4.2 Creating a new experiment • From the Home page click on Create a New Experiment. A blank experiment template will open in the Experim

622. Plate Layout Setup: a. From New b. Browse an existing template. c. Edit the selected template 3. Analysis Method Selection This chapter will

63 • Run information: The user can enter a user name and add any other comments relevant to the program. • Instrument settings: User-defined sett

64• 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 t

65 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

66• 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 (min

67change between these options. If the user is creating a new protocol and the instrument is not connected, it is not possible to select filter cartr

68• Click Add Ramp to open the ramp read parameters box: • Start Temperature: Starting temperature of the ramp can be anywhere from 4°C. • End T

69The read appears as a separate tab labelled according to the dye name. The temperature profile plot shows the reads as colour-coded points. Once th

7 Contents 1 Introduction ...

70 • 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

71• On the menu bar in Program Editor, select File and then Save As… • Change the file name if required. The file (.qprg format) will automat

723.4.4 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

73 • Show all wells: The well information table will show only the details for the currently selected sample type, e.g. standards

74• Calibrator (CAL): In relative quantification methods, the ratio of the DNA template in different samples may be normalized to

753.4.4.5 Replicates Choose how many replicates there are for each sample from the scroll-down menu. The Next group added to the plate

76 Undo: Cancels the previous operation. Rotate plate 180°: Useful if the plate was loaded into the instrument the opposite way round to the designa

773.4.4.8 Importing/exporting plate layouts from/to Microsoft® Excel Sample information can be copied into the Plate Layout Editor from an Excel

783.4.4.9 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 l

79 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 fil

81.16 Installing the filter cartridges ... 31 1.16.1 The role

803.4.5 Defining the analysis method 3.4.5.1 Selecting an analysis method The Analysis Selection box in the Experiment Editor (also found in the Re

81 • 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

82• Plus/minus scoring • Allelic discrimination • Multi-read See Chapter 4 for a specific discussion of each analysis method in terms of requireme

83 2. Passive reference dye (PRD) correction: • Check the box if this correction method is required (only available if a PRD was selected in the

84 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 la

85• Click Edit in the program or plate layout pane of the Experiment Editor and the appropriate editor will open. • Change settings in any step/st

863.5 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

873.5.1.3 Running an experiment 3.5.1.4 Run to report Unless otherwise informed, the system will wait for an acknowledgment from the user that t

88 • Save to the Experiment library folder if required (see section 3.4.6). Note that saving will over-write the previous version of th

89Experiment Editor and assign cartridges to each read. Alternately when the run button is pressed the following error window will appear: Selecting

9 3.3 Using Quansoft ... 55 3.3.1 Hom

903.5.2.2 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

91• Fluorescence graph: Data for a particular stage will be displayed in the graph. Choose to display readings for the entire plate or just for sele

92 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

933.7 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

94 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.

95results table as a strike-through). This is an important tool for selectively eliminating a well from the calculations. F. Results table: Contains

96name. This function can be turned off by clicking on the padlock icon. The user can also choose to save the experiment as just an exper

97 • If more sophisticated editing functions are required, access the Graph Properties… option from the Results Editor menu bar. Select Analysis an

98Clicking the PAR button brings up the analysis parameters relevant to the analysis being performed. The parameters can be edited

993.7.5 Log/Audit trail Clicking on this tab displays all the information about the run that may be required for GLP purposes. It i