TATA Motors CHECK ExpressPCR User Manual

Hide thumbs

page of 62

/ 62
Bookmarks

Quick Links

Download this manual

REAL-TIME PCR MADE SIMPLE!

USER GUIDE

Need help?

Do you have a question about the CHECK ExpressPCR and is the answer not in the manual?

Questions and answers

Summary of Contents for TATA Motors CHECK ExpressPCR

Page 1 REAL-TIME PCR MADE SIMPLE! USER GUIDE...
Page 2 WELCOME Thank you for choosing the Tata MD CHECK ExpressPCR. Tata MD CHECK ExpressPCR is a reliable, fast and compact qPCR platform for smart molecular testing labs. Novel Full Spectrum Optics deliver 120 optical channels of fluorescence data from every tube for exceptional multiplex PCR. High-...
Page 3 CONTENT This user guide will teach you what you need to know to start running your Tata MD CHECK ExpressPCR. It describes everything from connecting your instrument to the network to data analysis for your qPCR experiments. Contents include... SETUP ANALYSIS Learn how to install your Learn how to easily...
Page 4 SETUP INSTALLING YOUR EXPRESSPCR You should have the following items in the ExpressPCR flight case: 1. EXPRESSPCR INSTRUMENT 2. EXPRESSPCR USER GUIDE 3. POWER SUPPLY UNIT AND MAINS CABLE 4. ETHERNET CABLE 5. USB DRIVE Please keep the flight case and outer box the ExpressPCR system came in, in case you need to protect your ExpressPCR system during storage or shipment in future.
Page 5 The lid ensures that tubes are seated correctly in the wells, provides optical isolation, includes a heated lid to reduce condensation, and prevents dust falling into empty wells. USB PORT Insert the USB drive here to run an experiment from the USB drive.
Page 6 EXPRESSPCR CONNECTIONS The ExpressPCR has three connections. One in the front and two in back as shown below: Ethernet Port Power ETHERNET PORT POWER This is to connect your This is for connecting your ExpressPCR system to the ExpressPCR to your LAN provided power supply unit.
Page 7 POWERING ON YOUR EXPRESSPCR SYSTEM Place the ExpressPCR on your lab bench, and then connect AC power to the power supply unit. Your instrument will now turn on after a few seconds. The display LEDs will light up blue, and then turn green if a lid is closed or red if not.
Page 8 EXPRESSPCR CONNECTION MODES 1. Direct network connection 2. Local area network connection 3. USB drive connection...
Page 9 EXPRESSPCR HEATED LID The ExpressPCR heated lid will get hot. Please do not touch it. The heated lid will be preheated to 105°C if user activity is detected. This enables your run to start as soon as possible. After 5 minutes of inactivity the heated lid will be turned off to conserve energy.
Page 10 INSTALLING YOUR EXPRESSPCR SOFTWARE Your ExpressPCR USB Drive contains software for Windows, Mac OS X, and Linux operating systems. Please open the software file matching your chosen operating system. The latest version of the software can also be downloaded from our website www.tatamd.com WINDOWS MAC OS X...
Page 11 CONFIGURING YOUR EXPRESSPCR With your ExpressPCR software open, and your ExpressPCR system connected to the network, please connect to your ExpressPCR instrument from the software. Please select Configuration.
Page 12 Please select ExpressPCR. To add your new ExpressPCR System select the Add b utton. You will now be presented with a list of available instruments.
Page 13 Double-click on the instrument you wish to connect to, or pr ess Select with the instrument selected. The instrument will now be added to the list of Registered Instruments for use in the software.
Page 14 TIPS Here are some great tips to keep in mind whilst using your ExpressPCR instrument. 1. LID GETS WARM 2. DO NOT LEAVE THE LID OPEN 3. SPIN YOUR TUBES 4. REMOVE ALL BUBBLES 5. KEEP YOUR LAB CLEAN 6. KEEP YOUR INSTRUMENT CLEAN 7.
Page 15 REMOVE ALL BUBBLES Bubbles can cause optical artefacts as shown in the graph below. Ensure that no bubbles are present in reaction volumes. Artefact Cycle KEEP YOUR LAB CLEAN Please keep your work space clean including all lab equipment like surfaces, pipettes, and tube racks. This will keep the instrument clean and help maintain good results.
Page 16 EXPERIMENT This section will teach you everything you need to know to get started with ExpressPCR experiments. You will learn how to create, save, open, and close experiments. You will also learn how to set up an experiment, including thermal profile, sample information, and optical settings.
Page 17 EXPERIMENT SUMMARY Once you have created a new experiment the E xperiment Summary w ill be displayed as shown below: By default the experiment name will be “N ew Experiment” with a date and time stamp, which can be edited. The summary will give you information about the instrument you are...
Page 18 OPEN AN EXPERIMENT By selecting O pen y ou can open an experiment as shown below: FILES OF TYPE The Files of Type drop down menu gives you the option to open an experiment normally, or as a template that can be used for a new run.
Page 19 EXPERIMENT FILE An experiment file will contain all the data and analyses, but can not be re-run. To open as an Experiment File s elect the first option shown on the opposite page. EXPERIMENT FILE AS TEMPLATE Opening an experiment file as a template allows you to use the same profile, samples, settings, and analyses to re-...
Page 20 SAVING AN EXPERIMENT Once a run has finished it can either be automatically saved to a pre-set location on your computer, or manually saved by you. The experiment must be saved before the software is closed to ensure that data is not lost. AUTOMATIC SAVING To automatically save...
Page 21 MANUAL SAVING Once your run has finished you can save your experiment manually by clicking Save or Save As. The first time an experiment is saved, you will be prompted to select a location and filename to use. After this you can click Save to use the same location, overwriting the previous version, or Save As to choose a new location.
Page 22 SETTING UP A PROFILE In this section we will cover Hold Times, Programs and setting up a temperature profile. H O L D T I M E S During thermal cycling protocols hold times should be chosen to allow for the following: thermal equilibration of reaction volumes;...
Page 23 A thermal profile is required to perform an experiment. Select the profile tab as shown below: PROGRAMS To add a program to your experiment click A dd i n the P rograms p ane. Programs can be deleted by selecting them in the list then clicking D elete.
Page 24 TEMPERATURE PROFILE Once programs have been added to the experiment they will appear in the T emperature Profile p ane in the order they are present in the P rograms window as shown below: PROGRAM SETTINGS To alter the settings of a particular program you should first select it, then refer to the right hand...
Page 25 SAMPLES You will now learn how to set up samples and targets which can be defined as follows: SAMPLES TARGETS A description of the A molecular target e.g. specimen being analysed “x174”, detected with e.g. “Mouse #5” a specified fluorescent reporter e.g.
Page 26 ADDING SAMPLES AND TARGETS Samples and targets can be added and removed from the experiment by selecting + and - in the Samples and Targets pane. The order of samples and targets can be altered by clicking the Up and Down arrows in the Samples and Targets pane.
Page 27 DYE COMPATIBILITY The ExpressPCR system come pre-calibrated for over 20 different fluorescent labels, and a list of available dyes on the ExpressPCR system can be viewed below. Factory Calibrated SYBR Green I, ResoLight, Dyes FAM, VIC, HEX, Cy5, ROX, LC Green, SYTO 9, Cy5.5. Instrument Specific Calibration for (FAM, HEX, ROX, Cy5, Cy5.5.
Page 28 SAVING AND OPENING A SAMPLE SETUP If you wish to save or open sample and target information setup you can select Save As. . . , or Open, respectively. Sample and target setups can be saved in the following formats: Editable .csv files.
Page 29 RUN SETTINGS The ExpressPCR run settings tab is where you will determine the optical settings, view your dye calibrations and access the advanced settings. OPTICS SETTINGS The ExpressPCR will acquire optical data during the hold times. The longer the hold times are the better your optical data will be.
Page 30 STARTING A RUN FROM THE SOFTWARE To start a run from the ExpressPCR software select S tart Run. Y ou will then be presented with the auto save options (unless you have chosen not to be prompted) and then be asked to choose an instrument from the list of R egistered Instruments.
Page 31 LOADING STRIPS INTO YOUR EXPRESSPCR In order to ensure that the heated lid is balanced, please make sure that the mount contains at least a strip in rows A and D (as shown in blue below) or single tubes in wells A1, A8, D1 and D8. These positions can be filled with tubes containing reagents, or empty tubes.
Page 32 ANALYSIS This section will teach you about different types of analysis available. These can be grouped into the following groups. For information about more analysis options please contact technical support. QUANTIFICATION Determine accurate quantities of template or relative expression levels of genes using Absolute Quantification or R elative Quantification.
Page 33 SELECT ANALYSIS TYPE Once your experiment has completed you can add an analysis type by selecting the “+” button in the bottom left of the software window. Analysis types are separat ed int o Automatic and Manual sections. Automatic analysis types use automated data processing to provide accurate results with minimal user intervention.
Page 34 AUTOMATIC QUANTIFICATION POSITIVE / NEGATIVE CALLING The first step in amplification analysis is to determine which targets have produced a positive amplification curve and which amplifications have produced a negative amplification curve. For every amplification curve the software calculates three metrics, these metrics are used to determine which amplifications are positive.
Page 35 I N T E N S I T Y Intensity is a measure of the size of the exponential phase of the amplification. Note that this may be smaller for amplifications with a negative baseline drift. High P E R F O R M A N C E Performance is a measure related to the observed efficiency of amplification at later cycles.
Page 36 SETTINGS The software uses a combination of Quality (Q), Performance (P), and Intensity (I) thresholds to determine if an amplification is positive (green zone) or negative (red zone). All values (Q, P and I) must be above the relevant threshold for an amplification to be called positive.
Page 37 ARTEFACT FILTERING Biochemical and physical factors can cause fluorescence levels to change during a run. These are often observed during early cycles. Filtering out these artefacts can improve the accuracy of amplification analysis. Two settings enable you to control the process of filtering out such artefacts. E XC L .
Page 38 B AC KG R O U N D CO R R E C T I O N Background fluorescence, including drift, is estimated and removed. There are many potential sources of background fluorescence, e.g. emissions from unquenched probes. N O R M A L I SAT I O N Positive amplifications are rescaled to account for intensity variation between wells.
Page 39 RELATIVE QUANTIFICATION If you are performing relative quantification analysis then select R elative Quantification a t the top of the A uto Quant. pane. MULTIPLEXING The ExpressPCR supports multiplexing with a broad variety of dyes. F O R B E ST R E S U LTS : •...
Page 40 GENOTYPING AUTOMATIC ENDPOINT GENOTYPING TaqMan genotyping experiments can be analysed automatically using A utomatic Endpoint Genotyping. Genotypes of samples are determined by the ratio of endpoint fluorescence between two TaqMan probes. Thresholds for the fluorescence ratios are generated automatically as shown below: AUTOMATIC HIGH RESOLUTION MELTING A utomatic HRM c an be performed by using the A utomatic HRM m odule as shown on the following page:...
Page 41 D I F F E R E N C E , N O R M A L I Z E D A N D M E LT Once opened the analysis module will automatically determine optimised parameters and generate a set of D ifference, Normalized a nd M elt g raphs.
Page 42 MELTING AUTOMATIC TM CALLING Add an Automatic Tm Calling analyses to identify and characterise melting peaks. You can see the results below. Tm values will be presented in the table.
Page 43 AUTO SETTINGS The user has the option to change the target if the experiment contains more than one target. The signal-to-noise ratio setting determines how big a peak should be before it is called. Increasing this value will increase specificity of peak detection.
Page 44 RUN FUSION Run Fusion enables you to combine data from multiple runs from you ExpressPCR together for analysis. To fuse multiple experiments together hold down cmd on a Mac or Ctrl on a PC and select the files you wish to fuse from the open dialog window as shown below.
Page 45 If you are fusing experiments from multiple file locations you must select the first experiment and then select the “+” from the top left of the open file dialogue window as shown below. Now hold cmd/Ctrl and select the next experiment. Select Open to fuse your experiments together.
Page 46 EXPORT When you have finished analysing your data in the ExpressPCR software, you can export the results in a variety of ways, from raw data to user defined custom reports. This section will take you through the steps you need in order to do this. TABLE EXPORT From panes showing results in tabular format...
Page 47 To export data click the Export button, shown on the previous page. Data can be exported in the following formats: This is an editable data file that can be opened in many spreadsheet applications. This file format is suitable for archiving, printing and presentations, but cannot be edited.
Page 48 GRAPH EXPORT From panes showing results as graphs, such as amplification and melt curve data can be exported by selecting the button from the below example. The user can export information as bitmap images (.PNG), Scalable Vector Graphics (.SVG), Character-Separated Variable (.CSV), or Portable Document Format (.PDF).
Page 49 REPORT GENERATION Customisable reports can be generated from your experiment by selecting Experiment Report under the Experiment tab. This provides you with the ability to choose which parts of the data to include in a report. For example, items such as thermal profiles, sample information and various analysis results, e.g.
Page 50 MAINTENANCE This section will help you take care of your ExpressPCR. It covers the following areas: 1. CLEANING 3. ENVIRONMENTAL CONDITIONS 2. DISPOSABLES 4. DISASSEMBLY...
Page 51 CLEANING DAY-TO-DAY For day-to-day cleaning wipe the external surface of your instrument with a damp, soft, lint-free cloth. Then dry your instrument with another soft, lint-free cloth. Notes: Avoid abrasive cloths, towels, paper towels, and similar items that might cause damage. Before cleaning your instrument unplug all external power sources, devices, and cables.
Page 52 DECONTAMINATION If your instrument needs decontaminating please follow the instructions contained in the ExpressPCR Decontamination Guide. For details of the ExpressPCR Decontamination Guide please visit: www.tatamd.com Please note that for health and safety reasons you must print and complete a physical copy of the decontamination form, and include this with any instrument or lid that is returned.
Page 53 Disposables should have reproducible wall thicknesses, which are thin enough to ensure rapid temperature equilibration, but thick enough to avoid breakages. ENVIRONMENTAL CONDITIONS YOUR WORK SPACE Your ExpressPCR should be placed on a surface that is flat, dry, and not subject to draughts. Do not install your ExpressPCR instrument directly in the flow of air from an air conditioner or fan.
Page 54 ENVIRONMENTAL OPERATING CONDITIONS Humidity Temperature MAX: 80% at +32°C +15°C to +32°C MIN: 30% at +15 to +32°C Pressure 0 to 2000 MAMSL 80 to 106Kpa ENVIRONMENTAL STORING/ TRANSPORTING/PACKING CONDITIONS Humidity Temperature 10% to 95% -20°C to +60°C No condensation Pressure 0 to 3000 MAMSL 70 to 106Kpa...
Page 55 TROUBLESHOOTING This section will help you troubleshoot your ExpressPCR if you think something is wrong. Here are some frequently asked questions, and the answers. How do I create a All experiments can be used as a template. Click “Open” template? and then using the “file type”...
Page 56 Where is the threshold Modern methods of determining Cq values are not for determining Cq based on simple thresholds. values in Auto Quant? Modern methods of Cq determination are model based. Auto Quant fits a model of a PCR amplification to the fluorescence data observed.
Page 57 ERROR MESSAGES Your ExpressPCR will let you know when something is wrong by displaying an error. Most errors are reported as messages in the Status Bar in the ExpressPCR software. Some errors are reported by the instrument display LEDs. Status Bar.
Page 58 If you encounter an error, please make sure that the instrument is running in a lab within the specified environmental conditions, tubes have been loaded correctly, the lid has been fitted correctly, and all cables are attached correctly and securely. If the error still occurs, you may need to contact technical support with the following actions described below.
Page 59 c) Instrument is not yet ready to run. d) Instrument has finished a run and is waiting for tubes to be removed. e) Open lid, remove tubes and then add new ones. FAILURE This message will be followed by a short code. Please contact Technical Support.
Page 60 ERRORS REPORTED BY THE EXPRESSPCR DISPLAY LEDS Some errors are reported by the instrument display LEDs flashing red. If your instrument display LEDs are flashing red, please contact technical support. ERROR LOG FILES In the unlikely event that you encounter a problem the software can produce log files.
Page 61 NOTES All trademarks are the property of their respective owners. Design and specifications are subject to change without notice.
Page 62 www.tatamd.com Version 001 (EN)