abi 7500 Getting Started Manual
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abi 7500 Getting Started Manual

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Getting Started Guide
Applied Biosystems
7500/7500 Fast
Real-Time PCR System
Relative Standard Curve and
Comparative C
Experiments
T

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Summary of Contents for abi 7500

  • Page 1 Getting Started Guide Applied Biosystems 7500/7500 Fast Real-Time PCR System Relative Standard Curve and Comparative C Experiments...
  • Page 3 Getting Started Guide Get Started Applied Biosystems Design the Relative Standard 7500/7500 Fast Curve Experiment Real-Time PCR System Relative Standard Curve and Prepare the Relative Standard Comparative C Experiments Curve Reactions Run the Relative Standard Curve Experiment Analyze the Relative Standard...
  • Page 4 NOTICE TO PURCHASER: Label License The 7500/7500 Fast Real-Time PCR Systems are covered by US p atents and corresponding claims in their non-US counterparts, owned by Appl ied Biosystems . No right is conveyed expressly, by implication, or by estoppel under any other patent claim, such as claims to apparatus, reagents, kits, or methods such as 5 nuclease methods.

  • Page 5: Table Of Contents

    About the 7500/7500 Fast System ........
  • Page 6 Define the Experiment Properties ........114 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve...
  • Page 7 Publish the Data ........... . . 202 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve...
  • Page 8 Index ......... 229 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve...

  • Page 9: Preface

    Preface How to Use This Guide About the System The guides listed below are shipped with the Applied Biosystems 7500 and 7500 Fast Documentation Real-Time PCR Systems. Guide Purpose and Audience Applied Biosystems 7500/7500 Fast Real- Explains how to perform experiments on the 7500/7500 Fast...
  • Page 10 • Know how to handle DNA and/or RNA samples and prepare them for PCR. • Understand data storage, file transfer, and copying and pasting. • Have networking experience, if you plan to integrate the 7500/7500 Fast system into your existing laboratory data flow.

  • Page 11: How To Obtain More Information

    Software Version 3.0 Getting Started Guide 4362460 ® TaqMan Gene Expression Assays Protocol 4333458 User Bulletin #2: Relative Quantitation of Gene Expression 4303859 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 12 Endogenous Control Assays to Select an Endogenous Control 127AP08 for Experimental Studies Application Note For more documentation, see “How to Obtain Support” on page xi. Note: Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 13: How To Obtain Support

    Preface How to Obtain Support Obtaining The 7500 Software Help describes how to use each feature of the touchscreen. Open the Information from Help from within the software by doing one of the following: the Software Help • Press F1.

  • Page 14: Safety Conventions Used In This Document

    ELECTRICAL HAZARD. Failure to ground the instrument properly can lead to an electrical shock. Ground the instrument according to the provided instructions. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 15: Symbols On Instruments

    Indicates the presence of a laser inside the instrument and to proceed with appropriate caution. Indicates the presence of moving parts and to proceed with appropriate caution. xiii Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 16: Safety Labels On Instruments

    AVERTISSEMENT Cet instrument est conçu 12 V, 75 W Halogen lamps only. pour des lampes d'halogène de 12 V et 75 W seulement. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 17 The Applied Biosystems 7500/7500 Fast Real-Time PCR System contains warnings at Warnings the locations shown below. Attention Physical hazard Physical hazard Attention Attention Attention Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 18: General Instrument Safety

    Before using a cleaning or decontamination method other than those Decontaminating recommended by the manufacturer, verify with the manufacturer that the proposed the Instrument method will not damage the equipment Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 19: Chemical Safety

    • Save Target As – To download a PDF version of the document to a destination that you choose For the MSDSs of chemicals not distributed by Applied Biosystems, contact the Note: chemical manufacturer. xvii Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 20 MSDS. • Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. xviii Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 21: Chemical Waste Safety

    Radioactive or biohazardous materials may require special handling, and IMPORTANT! disposal limitations may apply. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 22: Electrical Safety

    PHYSICAL INJURY HAZARD. Moving parts can crush and cut. Keep hands clear of moving parts while operating the instrument. Disconnect power before servicing the instrument. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 23: Biological Hazard Safety

    • Position the keyboard, mouse, and monitor to promote relaxed body and head postures. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 24: Safety And Electromagnetic Compatibility (Emc) Standards

    This instrument has been tested to and complies with standard AS/NZS 2064, “Limits Standards and Methods Measurement of Electromagnetic Disturbance Characteristics of Industrial, Scientific, and Medical (ISM) Radio-frequency Equipment.” xxii Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 25: Get Started

    About the 7500/7500 Fast System ........

  • Page 26: About The 7500/7500 Fast System

    • After the PCR. Regardless of the run type, a data collection point or read on the 7500/7500 Fast instrument consists of three phases: 1. Excitation – The instrument illuminates all wells of the reaction plate within the instrument, exciting the fluorophores in each reaction.
  • Page 27 • Standard curve experiments – Refer to the Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve and Experiments. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 28: Supported Consumables

    Optical Tube without Cap ™ ™ MicroAmp Optical 8-Cap Strip, 0.2-mL MicroAmp Optical Adhesive Film ® MicroAmp Reaction Tube with Cap, 0.2-mL Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 29 ® Microamp Splash Free Support Base ™ MicroAmp Optical 8-Cap Strip ™ MicroAmp Fast 8-Tube Strip ™ MicroAmp Optical Adhesive Film Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 30 (PN 4306737) Notch at top-right corner by A12 Notch at top-left corner by A1 30-µL 100-µL maximum maximum reaction reaction volume volume Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 31: About Relative Standard Curve And Comparative C Experiments

    Housekeeping genes can be used as endogenous controls. • Replicates – The total number of identical reactions containing identical samples, components, and volumes. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 32 • Negative Controls – Wells that contain water or buffer instead of sample template. No amplification of the target should occur in negative control wells. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 33 Applied Biosystems recommends that you determine that the PCR efficiencies for the target assay and the endogenous control assay are approximately equal. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 34 UNG enzyme can be used to prevent carryover contamination. ® For more information on AmpErase UNG, refer to the Real Time PCR System Note: Reagent Guide. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 35 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 36: How To Use This Guide

    This guide functions as both a tutorial and as a guide for performing your own experiments. Using This Guide Using the example experiment data provided with the 7500 software, you can use this as a Tutorial guide as a tutorial for performing a relative standard curve or comparative C experiment on a 7500/7500 Fast system.
  • Page 37 Chapters 2 through 9 includes a set of guidelines that you can use to perform your own experiments. Additionally, you can use one of the other workflows provided in the 7500 software to perform your experiments. The table below summarizes the available workflows.

  • Page 38: About The Example Experiments

    – For the endogenous control assay (GAPDH), the assay ID is Hs99999905_m1 (RefSeq NM_002046.3). Reaction Plate Layout The 7500 software displays the 96-well plate layout, as shown below. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 39 – For the endogenous control assay (18S), the 18S Assay Mix: Hs99999901_s1. Reaction Plate Layout The 7500 software displays the 96-well plate layout as shown below. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 40 • In Chapter 5, you view results in a relative standard curve example experiment file that contains run data. The data file for the example experiments is installed with the 7500 software. You can find the data file for the example experiment on your computer at: <drive>:\Applied Biosystems\<software name>\experiments\...

  • Page 41: Example Experiment Workflow

    4. Monitor the run. 4. Monitor the run. 5. Unload the instrument. 5. Unload the instrument. (continued on page 18) (continued on page 18) Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 42 5. View the experiment data. 6. View the Multicomponent Data. 7. View multiple plots. 8. View the quality summary. 9. Publish the data. End Experiment Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 43: Design The Relative Standard Curve Experiment

    Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 44: Chapter Overview

    Chapter Overview Chapter Overview This chapter explains how to use the Design Wizard in the 7500 software to set up the relative standard curve example experiment. The Design Wizard guides you through Applied Biosystems recommended best practices as you enter design parameters for the example experiment.

  • Page 45: Create A New Experiment

    <software name> is the current version of the 7500 software. 2. In the Home screen, click Design Wizard to open the Design Wizard. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 46: Define The Experiment Properties

    In the relative standard curve example experiment: Example • The experiment is identified as an example. Experiment • The instrument that is selected to run the experiment is the 7500 instrument. ® • A MicroAmp Optical 96-Well Reaction Plate is used.
  • Page 47 • Consumables – See “Supported Consumables” on page 4. • Quantitation experiments – Refer to the Real Time PCR System Reagent Guide. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 48: Define The Methods And Materials

    3. Select Standard (~ 2 hours to complete a run) for the ramp speed. 4. Select cDNA (complementary DNA) for the template type. 5. Click Next. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 49 • Select the appropriate ramp speed for the instrument run: IMPORTANT! Select a Fast run only if you use a 7500 Fast system. Fast runs cannot be performed on a 7500 system. – Select Fast (~ 40 minutes to complete a run) if you are using Fast reagents for the PCR reactions.

  • Page 50: Set Up The Targets

    Set Up the Targets For More For more information on: Information • Completing the Methods and Materials screen – Open the 7500 Software Help by clicking or pressing F1. • Using Advanced Setup – See “Advanced Setup Workflow” on page 204.
  • Page 51 For all targets, leave blank the (Optional) Enter Gene Name field. You can search for the gene/assay ID when you order your materials (see “Order Materials for the Experiment” on page 43). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 52 • Selecting an endogenous control – See the Application Note Using TaqMan Endogenous Control Assays to Select an Endogenous Control for Experimental Studies. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 53: Set Up The Standards

    5. Review the Standard Curve Preview pane for each assay. The standard curves have the following points: 200, 20, 2, 0.2, and 0.02. 6. Click Next. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 54 If your starting quantity is the lowest quantity, select a concentration factor such as 2✕, 3✕, and so on. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 55: Set Up The Samples

    In the Color field, leave the default. 6. Select No Biological Replicates. 7. Select All Sample/Target Reactions to test all targets in all samples. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 56 Inclusion of biological replicates can give insight into any natural variation that is present within the population. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 57 • Completing the Samples screen – Open the 7500 Software Help by clicking pressing F1. • Using Advanced Setup – See “Advanced Setup Workflow” on page 204. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 58: Set Up The Relative Quantitation Settings

    • Reference samples (also known as calibrators) and endogenous controls – Refer to User Bulletin #2: Relative Quantitation of Gene Expression (PN 4303859B). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 59: Set Up The Run Method

    3. Make sure the thermal profile displays the holding and cycling stages shown below. If necessary, click a temperature or time setting, then change it. 4. Click Next. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 60: Review The Reaction Setup

    When you design your own relative standard curve experiment: Guidelines • Enter a number from 10 to 100 for the reaction volume/well. The 7500 system supports reaction volumes from 20 to 100 µL. The 7500 Fast system supports reaction volumes from 10 to 30 µL.
  • Page 61 Sample (10✕) or Standard 17.50 Total Volume 50.0 ‡ The sample or standard volume is limited to 10% of the total reaction volume. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 62 6. In the Reactions for GAPDH pane: Make sure the Master Mix Concentration field displays 2.0✕. Make sure the Assay Mix Concentration field displays 20.0✕. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 63 Dilution 2 2.02 18.15 20.17 4 (0.2) Dilution 3 2.02 18.15 20.17 0.04 5 (0.02) Dilution 4 2.02 18.15 20.17 0.004 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 64 Total Volume of Diluted Sample Sample Name (ng/µL) (µL) (µL) (µL) Liver 100.0 17.00 17.00 34.00 Kidney 100.0 17.00 17.00 34.00 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 65 2. In the Print Reaction Setup Instructions dialog box, select: • Detailed Reaction Setup Instructions • Include Plate Layout • Use sample color 3. Click Print. 4. Click Next. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 66 – Select Custom if you are designing your own assays with Primer Express software. • Enter a number from 10 to 100 for the reaction volume/well. The 7500 system supports reaction volumes from 20 to 100 µL. The 7500 Fast system supports reaction volumes from 10 to 30 µL.

  • Page 67: Order Materials For The Experiment

    In the Find Assay Results dialog box, select the Hs00153408_m1 row, then click Apply Assay Selection. Click the Enter Gene Name field, enter GAPDH, then click Find Assay. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 68 Online ordering through the Applied Biosystems Store is not available in all countries. Contact your local Applied Biosystems representative for help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 69 When you are connected to the Applied Biosystems Store, follow the prompts to complete your order. 6. Go to “Finish the Design Wizard” on page 46. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 70: Finish The Design Wizard

    Finish the Design Wizard Finish the Design Wizard, review the plate layout, then select an exit option. About the The 7500 software automatically selects locations for the wells in the reaction plate. In Example the relative standard curve example experiment: Experiment •...
  • Page 71 By default, the example experiment is saved to the Note: <drive>:\Applied Biosystems\<software name>\experiments folder. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 72 In the Data Folder field, browse to then select the desired location. For More For more information on using Advanced Setup, see “Advanced Setup Workflow” on Information page 204. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 73: Prepare The Relative Standard Curve Reactions

    Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 74: Chapter Overview

    4. Prepare the reaction mix for each target assay. 5. Prepare the reaction plate. Run the Experiment (Chapter 4) Analyze the Experiment (Chapter 5) End Experiment Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 75: Prepare The Template

    AM9935 Pack ™ DNAZap AM9890 Choice of RNA sample preparation products, as listed See above above under the qRT-PCR Starter Pack. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 76 Wear appropriate protective eyewear, clothing, and gloves. 2. Prepare the cDNA reactions. 3. Perform reverse transcription on a thermal cycler. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 77: Prepare The Sample Dilutions

    Prepare the Sample Dilutions Perform sample dilutions before adding the samples to the final reaction mix. Dilute the samples using the volumes that were calculated by the 7500 software (“To complete the Sample Dilution Calculations tab:” on page 40). About the...
  • Page 78 For more information on Applied Biosystems assays, refer to the: Information ® • TaqMan Gene Expression Assays Protocol ® • Custom TaqMan Gene Expression Assays Protocol. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 79: Prepare The Standard Dilution Series

    Prepare the Standard Dilution Series Prepare the Standard Dilution Series You prepare the standard dilution series using the volumes that were calculated by the 7500 software (“To complete the Reaction Mix Calculations tab for the c-myc assay” on page 37 and “To complete the Reaction Mix Calculations tab for the GAPDH assay”...
  • Page 80 Series for the • GAPDH Std. 2 GAPDH Assay • GAPDH Std. 3 • GAPDH Std. 4 • GAPDH Std. 5 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 81 • The quality of pipettors and tips and the care used in measuring and mixing dilutions affect accuracy. • Use TE buffer or water to dilute the standards. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 82: Prepare The Reaction Mix

    You prepare the reaction mix using the components and volumes that were calculated by the 7500 software (“To complete the Reaction Mix Calculations tab for the c-myc assay” on page 37 and “To complete the Reaction Mix Calculations tab for the GAPDH assay”...
  • Page 83 5. Centrifuge the tubes briefly to remove air bubbles. 6. Place the reaction mixes on ice until you prepare the reaction plate. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 84: Prepare The Reaction Plate

    – 30 Standard wells – 6 Negative control wells – 48 Empty wells • The plate layout that is automatically generated by the 7500 software is used: Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve...
  • Page 85 Centrifuge the tube briefly to remove air bubbles. Add 50 µL of the negative control reaction to the appropriate wells in the reaction plate. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 86 Add 50 µL of the unknown (sample) reaction to the appropriate wells in the reaction plate. 4. Seal the reaction plate with optical adhesive film. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 87 Haziness disappears when the film comes into contact with the heated cover in the instrument. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 88 • Consumables – See “Supported Consumables” on page 4. • Using Advanced Setup to change the plate layout – See “Advanced Setup Workflow” on page 204. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 89: Run The Relative Standard Curve Experiment

    Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 90: Chapter Overview

    Chapter 4 Run the Relative Standard Curve Experiment Chapter Overview Chapter Overview This chapter explains how to perform a run on the Applied Biosystems 7500/7500 Fast Real-Time PCR System. Example Start Relative Standard Curve Experiment Experiment Workflow Design the Experiment (Chapter 2)

  • Page 91: Prepare For The Run

    3. In the Open dialog box, navigate to the experiments folder (default) at: <drive>:\Applied Biosystems\<software name>\experiments 4. Double-click the example experiment file that you created in Chapter 2. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 92 Fast Reaction 8-tube strips can be used only on the 7500 Fast system. ® For the 7500 system, use MicroAmp Optical 8-Tube Strips. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 93: Enable The Notification Settings (Optional)

    • Place tube strips with samples in the PPH vertically, starting in columns 6 and 7 and moving outward. • A maximum of 6 tube strips can be used in the 7500 Fast system. Leave columns 2, 3, 10, and 11 empty.
  • Page 94 7. Set the authentication settings: Select Yes for Server requires authentication. In the User Name field, enter Example User. In the Password field, enter password. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 95: Start The Run

    Run Guidelines When you set up the 7500/7500 Fast system for automatic notification: • Your system must be set up for network use. Refer to the Applied Biosystems 7500/7500 Fast Real-Time PCR System Maintenance Guide. • Select the events for which you want to receive e-mail notifications. Select: –...

  • Page 96: Monitor The Run

    To view data in the Amplification Plot screen, select the wells that you want to view in the View Plate Layout tab. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 97 Change the number of cycles In the Number of Cycles field, enter a number. Apply your changes Click Save Run Method. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 98: Unload The Instrument

    2. Remove the calibration plate. 3. Push the tray door to close it. GR2477 7300/7500 barcode on plate GR2471 7500 tray top view Push Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 99: Analyze The Relative Standard Curve Experiment

    Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 100: Chapter Overview

    2. View the quality summary. 3. Omit wells. 4. View the multicomponent plot. 5. View the raw data plot. End Experiment Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 101: Section 5.1: Review Results

    Publish the Data ........... 97 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C...

  • Page 102: Analyze The Experiment

    For the relative standard curve example experiment, use the data file that is installed with Example the 7500 software. The data file was created with the same design parameters that are Experiment provided in Chapter 2, then run and analyzed on a 7500/7500 Fast instrument.
  • Page 103 2. Right-click well A9, then select Include. 3. Click Analyze. The software analyzes the data using the default analysis settings. outlier Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 104 Guidelines When you analyze your own relative standard curve experiment: • Immediately after a run, the 7500 software automatically analyzes the data using the default analysis settings, then displays the Amplification Plot screen on your computer. • To reanalyze the data, select all the wells in the plate layout, then click Analyze.
  • Page 105 (Show plots in two columns). 5. To display a specific plot, select the plot in the drop-down list above each plot display. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 106: View The Standard Curve

    Amplification Target Slope Y-Inter Value Efficiency (Eff%) GAPDH -3.506 27.226 0.999 92.853 c-myc -3.696 23.82 0.998 86.438 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 107 Look at the values in the C column. In the example experiment, the C values are within the expected range (>8 and <35). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 108 • The Standard Curve screen – Open the 7500 Software Help by clicking pressing F1. ® • Amplification efficiency – Refer to the Amplification Efficiency of TaqMan Gene Expression Assays Application Note. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 109: View The Amplification Plot

    In the relative standard curve example experiment, you review each target in the Example Amplification Plot for: Experiment • Correct baseline and threshold values • Outliers Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 110 (Show a legend for the plot). This is a toggle button. When the legend is displayed, the button changes Note: to Hide the plot legend. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 111 Verify that the baseline is set correctly: The end cycle should be set a few cycles before the cycle number where significant fluorescence is detected. In the example experiment, the baseline is set correctly. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 112 Select the Threshold check box to show the threshold (default). Verify that the threshold is set correctly. In the example experiment, the threshold is in the exponential phase. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 113 (well A9). You omit this well in the troubleshooting section (“Omit Wells from the Analysis” on page 105). outlier Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 114 • Correct baseline and threshold values – See “Threshold Examples” on page 91 and “Baseline Examples” on page 92. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 115 Drag the threshold bar down into the exponential phase of the curve. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 116 • Omit wells (see “Omit Wells from the Analysis” on page 105). • Manually adjust the baseline and/or threshold (see “View the Analysis Settings” on page 100). For More For more information on the Amplification Plot screen, open the 7500 Software Help by Information clicking or pressing F1.

  • Page 117: View The Gene Expression Plot And Well Table

    • The sample name, target name, task, and dyes • The calculated threshold cycle (C ), normalized fluorescence (Rn), and quantity values • Flags Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 118 Log10 graph type, the expression level of the reference sample appears as 0 in the graph (log10 of 1 = 0). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 119 Note: The maximum includes the variability associated with the endogenous control and targets in only the test samples. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 120 For more information on the Gene Expression Plot screen or Well Table, open the Information 7500 Software Help by clicking or pressing F1. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 121: Publish The Data

    • Export data to applications such as Microsoft Excel and Microsoft PowerPoint For information on performing these tasks, open the 7500 Software Help by clicking or pressing F1. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 122 Chapter 5 Analyze the Relative Standard Curve Experiment Publish the Data Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 123: Section 5.2: Troubleshoot (If Needed)

    View the Raw Data Plot ..........109 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C...

  • Page 124: View The Analysis Settings

    The Analysis Settings dialog box displays the analysis settings for the threshold cycle ), flags, and advanced options. If the default analysis settings in the 7500 software are not suitable for your experiment, you can change the settings, then reanalyze your experiment.
  • Page 125 Analysis Unless you have already determined the optimal settings for your experiment, use the Guidelines default analysis settings in the 7500 software. If the default settings are not suitable for your experiment, you can change the: • C Settings – Use this tab to manually set the threshold and baseline. When manually...
  • Page 126 3. In the Select Multiple Endogenous Controls dialog box, select the targets that you want to use as endogenous controls, then click OK. ‘ For More For more information on the analysis settings, open the 7500 Software Help by pressing Information F1 when the Analysis Settings dialog box is open. Notes...

  • Page 127: View The Qc Summary

    Chapter 5 Analyze the Relative Standard Curve Experiment View the QC Summary View the QC Summary The QC Summary screen displays a list of the 7500 software flags, and includes the flag frequency and location for the open experiment. About the...
  • Page 128 For more information on the QC Summary screen or on flag settings, open the Information 7500 Software Help by clicking or pressing F1. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 129: Omit Wells From The Analysis

    Look for any outliers in the replicate group (be sure they are flagged). In the example experiment, well A9 is the outlier. Select the Omit check box next to well A9. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 130 If needed, remove outliers manually using the Well Table. See “Omit Wells” on page 105 to remove the outliers in your experiment. For More For more information on omitting wells from the analysis, open the 7500 Software Help Information by clicking or pressing F1.

  • Page 131: View The Multicomponent Plot

    5. View the FAM dye signal. In the example experiment, the FAM dye signal increases throughout the PCR process, which indicates normal amplification. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 132 7. Select the negative control wells one at a time and check for amplification. In the example experiment, no amplification occurs in the negative control wells. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 133: View The Raw Data Plot

    The legend displays the color code for each row of the reaction plate. In the Note: example shown below, Row A is red, Row B is yellow/green, Row C is green, and so on. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 134 Guidelines each filter: • Characteristic signal growth • No abrupt changes or dips For More For more information on the Raw Data Plot screen, open the 7500 Software Help by Information clicking or pressing F1. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve...

  • Page 135: Design The Comparative C Experiment

    Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 136: Chapter Overview

    Chapter 6 Design the Comparative C Experiment Chapter Overview Chapter Overview This chapter explains how to use the Design Wizard in the 7500 software to set up the comparative C (∆∆C ) example experiment. The Design Wizard guides you through Applied Biosystems recommended best practices as you enter design parameters for the example experiment.

  • Page 137: Create A New Experiment

    <software name> is the current version of the 7500 software. 2. In the Home screen, click Design Wizard to open the Design Wizard. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 138: Define The Experiment Properties

    4. Click the Comments field, then enter Example Experiment for the Comparative Ct Getting Started Guide. 5. Select 7500 (96 Wells). 6. Select Quantitation for the experiment type. 7. Click Next. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 139 • Consumables – See “Supported Consumables” on page 4. • Quantitation experiments – Refer to the Real Time PCR System Reagent Guide. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 140: Define The Methods And Materials

    3. Select Standard (~ 2 hours to complete a run) for the ramp speed. 4. Select cDNA (complementary DNA) for the template type. (default) 5. Click Next. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 141 7500/7500 Fast system, you must design your experiment using Advanced Setup instead of the Design Wizard. Note: Fast runs can be performed only on the 7500 Fast system and an upgraded 7500 system. • Select the appropriate ramp speed for the instrument run: –...
  • Page 142 You are adding purified genomic DNA to the PCR reactions. For More For more information on: Information • Completing the Methods and Materials screen – Open the 7500 Software Help by clicking or pressing F1. • Determining PCR efficiencies – Open the 7500 Software Help by clicking pressing F1.

  • Page 143: Set Up The Targets

    For all targets, leave blank the (Optional) Enter Gene Name field. You can search Note: for the gene/assay ID when you order your materials (see “Order Materials for the Experiment” on page 131). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 144 • Selecting an endogenous control – See the Application Note Using TaqMan Endogenous Control Assays to Select an Endogenous Control for Experimental Studies. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 145: Set Up The Samples

    Universal (Use default color) 6. Select No Biological Replicates. 7. Select All Sample/Target Reactions to test all targets in all samples. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 146 • Select which targets to test in the samples: – Select All Sample/Target Reactions to test all targets in all samples. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 147 • Completing the Samples screen – Open the 7500 Software Help by clicking pressing F1. • Using Advanced Setup – See “Advanced Setup Workflow” on page 204. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 148: Set Up The Relative Quantitation Settings

    F1. • Reference samples (also known as calibrators) and endogenous controls – See User Bulletin #2: Relative Quantitation of Gene Expression. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 149: Set Up The Run Method

    3. Make sure the thermal profile displays the holding and cycling stages shown below. If necessary, add click on a temperature or time setting and change it. 4. Click Next. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 150 When you design your own comparative C experiment: Guidelines • Enter a number from 10 to 100 for the reaction volume/well. The 7500 system supports reaction volumes from 20 to 100 µL. The 7500 Fast system supports reaction volumes from 10 to 30 µL.

  • Page 151: Review The Reaction Setup

    6. In the Reactions for 18S pane: Make sure the Master Mix Concentration field displays 2.0✕. Make sure the Assay Mix Concentration field displays 20.0✕. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 152 2. Click the Diluted Sample Concentration (10✕ for Reaction Mix) field, then enter 50.0. 3. In the unit drop-down list, select ng/µL (default). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 153 • Detailed Reaction Setup Instructions • Include Plate Layout • Use sample color 3. Click Print to print the reaction setup instructions. 4. Click Next. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 154 – Select Custom if you are designing your own assays with Primer Express software. • Enter a number from 10 to 100 for the reaction volume/well. The 7500 system supports reaction volumes from 20 to 100 µL. The 7500 Fast system supports reaction volumes from 10 to 30 µL.

  • Page 155: Order Materials For The Experiment

    • EGR3 Assay Mix: Hs00231780_m1 • MAOB Assay Mix: Hs00168533_m1 • OGDH Assay Mix: Mm00803121_m1 • OSGEP Assay Mix: Hs00215099_m1 • SERPING1 Assay Mix: Hs00163781_m1 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 156 Online ordering through the Applied Biosystems Store is not available in all countries. Contact your local Applied Biosystems representative for help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 157 When you are connected to the Applied Biosystems Store, follow the prompts to complete your order. 7. Go to “Finish the Design Wizard” on page 134. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 158: Finish The Design Wizard

    Finish the Design Wizard To finish the Design Wizard, review the plate layout, then select an exit option. About the The 7500 software automatically selects locations for the wells in the reaction plate. In Example the comparative C example experiment: Experiment •...
  • Page 159 If the plate layout is incorrect, click Return to the Wizard, then check your Note: entered values. 3. Click Save Experiment. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 160 In the Data Folder field, browse to then select the desired location. For More For more information on using Advanced Setup, see “Advanced Setup Workflow” on Information page 204. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 161: Prepare The Comparative C

    Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 162: T Reactions

    3. Prepare the reaction mix for each target assay. 4. Prepare the reaction plate. Run the Experiment (Chapter 8) Analyze the Experiment (Chapter 9) End Experiment Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 163: Prepare The Template

    Recommended if you are using SYBR Green reagents. If you are using SYBR Green reagents, use TURBO DNA-free in conjunction with the RiboPure Kit. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 164 Wear appropriate protective eyewear, clothing, and gloves. 2. Prepare the cDNA reactions. 3. Perform reverse transcription on a thermal cycler. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 165 • Preparing RNA or gDNA templates – Refer to the protocol for the purification reagents that you select. To locate Applied Biosystems purification reagents, visit: http://www.appliedbiosystems.com/ Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 166: Prepare The Sample Dilutions

    Prepare the Sample Dilutions Perform sample dilutions before adding the samples to the final reaction mix. Dilute the samples using the volumes that were calculated by the 7500 software (“Select the Sample Dilution Calculations tab.” on page 128). About the...
  • Page 167 For more information on Applied Biosystems assays, refer to the: Information ® • TaqMan Gene Expression Assays Protocol ® • Custom TaqMan Gene Expression Assays Protocol. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 168: Prepare The Reaction Mix

    Prepare the reaction mix using the components and volumes that were calculated by the 7500 software (“Complete the Reaction Mix Calculations Tab for the Assays” on page 127). The software calculates the volumes for all components for the PCR reactions.
  • Page 169 – Thaw any frozen samples by placing them on ice. When thawed, resuspend the samples by vortexing, then centrifuge the tubes briefly. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 170: Prepare The Reaction Plate

    • The reaction volume is 50 µL/well. • The reaction plate contains: – 96 Unknown wells – 0 Negative Control wells – 0 Empty wells • The plate layout that is automatically generated by the 7500 software is used: Required • Microcentrifuge tubes Materials • Pipettors •...
  • Page 171 Centrifuge the tubes briefly to remove air bubbles. Add 50 µL of the unknown (sample) reaction to the appropriate wells in the reaction plate. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 172 Haziness disappears when the film comes into contact with the heated cover in the instrument. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 173 • Consumables – See “Supported Consumables” on page 4. • Using Advanced Setup to change the plate layout – See “Advanced Setup Workflow” on page 204. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 174 Chapter 7 Prepare the Comparative C Reactions Prepare the Reaction Plate Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 175: Run The Comparative C Experiment

    Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 176: Chapter Overview

    Chapter 8 Run the Comparative C Experiment Chapter Overview Chapter Overview This chapter explains how to perform a run on the Applied Biosystems 7500/7500 Fast Real-Time PCR Systems. Example Start Comparative C (∆∆C ) Experiment Experiment Workflow Design the Experiment (Chapter 6)

  • Page 177: Prepare For The Run

    3. In the Open dialog box, navigate to the experiments folder (default) at: <drive>:\Applied Biosystems\<software name>\experiments 4. Double-click the example experiment file that you created in Chapter 6. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 178: Run The Experiment

    • “Start the Run” on page 71 • “Monitor the Run” on page 72 • “Unload the Instrument” on page 74 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 179: Analyze The Comparative C Experiment

    Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 180: Chapter Overview

    5. View the experiment data. 6. View the Multicomponent Data. 7. View multiple plots. 8. View the quality summary. 9. Publish the data. End Experiment Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 181: Section 9.1: Review Results

    Publish the Data ..........174 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C...

  • Page 182: Analyze The Experiment

    Example 7500 software. This data file was created with the same design parameters that are Experiment provided in Chapter 6, then run and analyzed on a 7500/7500 Fast instrument.
  • Page 183 Guidelines When you analyze your own comparative C experiment: • Immediately after a run, the 7500 software automatically analyzes the data using the default analysis settings, then displays the Amplification Plot screen on your computer. • To reanalyze the data, select all the wells in the plate layout, then click Analyze.
  • Page 184 Shift+click the desired wells in the plate layout. 4. To select all 96 wells, click the upper left corner of the plate layout. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 185 (Show plots in two columns). 5. To display a specific plot, select the plot in the drop-down list above each plot display. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 186: View The Gene Expression Plot And Well Table

    • The Well Table to evaluate the precision of the replicate groups. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 187 Log10 graph type, the expression level of the reference sample appears as 0 in the graph (log10 of 1 = 0). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 188 Analysis Settings dialog box. Note: The maximum includes the variability associated with the endogenous control and targets in only the test samples. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 189 For more information on the Gene Expression Plot screen or Well Table, open the Information 7500 Software Help by clicking or pressing F1. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 190: View The Amplification Plot

    Amplification Example Plot for: Experiment • Correct baseline and threshold values • Outliers Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 191 (Show a legend for the plot). Note: This is a toggle button. When the legend is displayed, the button changes to Hide the plot legend. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 192 Consequently, the plot overlays the baseline and threshold labels for all targets until you select an individual target from the Target drop-down list. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 193 Verify that the threshold is set correctly. The example experiment contains multiple thresholds, one for each target assay. The thresholds for all targets occur in the exponential phase. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 194 7. Repeat steps 2 through 6 for the EGR3, MAOB, OGDH, OSGEP, and SERPING1 wells. In the example experiment, there are no outliers for any of the targets. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 195 • Correct baseline and threshold values – See the “Threshold Examples” on page 172 and “Baseline Examples” on page 173. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 196 Drag the threshold bar down into the exponential phase of the curve. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 197 • Omit wells (see “Omit Wells from the Analysis” on page 181). • Manually adjust the baseline and/or threshold (see “View the Analysis Settings” on page 176). For More For more information on the Amplification Plot screen, open the 7500 Software Help by Information clicking or pressing F1.

  • Page 198: Publish The Data

    • Export data to applications such as Microsoft Excel and Microsoft PowerPoint For information on performing these procedures, open the 7500 Software Help by clicking or pressing F1. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 199: Section 9.2: Troubleshoot (If Needed)

    View the Raw Data Plot ..........185 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C...

  • Page 200: View The Analysis Settings

    The Analysis Settings dialog box displays the analysis settings for the threshold cycle ), flags, relative quantitation, and advanced options. If the default analysis settings in the 7500 software are not suitable for your experiment, you can change the settings in the Analysis Settings dialog box, then reanalyze your experiment.
  • Page 201 Analysis Unless you have already determined the optimal settings for your experiment, use the Guidelines default analysis settings in the 7500 software. If the default settings are not suitable for your experiment, you can change the: • C Settings – Use this tab to manually set the threshold and baseline. When...
  • Page 202 To set assay efficiency, click 100.0 in the Efficiency (%) column of an assay, then type a percentage value between 1% and 100%. For More For more information on the analysis settings, open the 7500 Software Help by pressing Information F1 when the Analysis Settings dialog box is open.

  • Page 203: View The Qc Summary

    Chapter 9 Analyze the Comparative C Experiment View the QC Summary View the QC Summary The QC Summary screen displays a list of the 7500 software flags, and includes the flag frequency and location for the open experiment. About the In the comparative C...
  • Page 204 For more information on the QC Summary screen or on flag settings, open the Information 7500 Software Help by clicking or pressing F1. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 205: Omit Wells From The Analysis

    Look for any outliers in the replicate group (be sure they are flagged). In the example experiment, there are no outliers. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 206: View The Multicomponent Plot

    5. Click Analyze to reanalyze the experiment data with the outlying well(s) removed from the analysis. For More For more information on omitting wells from the analysis, open the 7500 Software Help Information by clicking or pressing F1. Within the Help, search for the omit well topics: 1.
  • Page 207 6. View the ROX dye signal. In the example experiment, the ROX dye signal is constant throughout the PCR process, which indicates typical data. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 208 For More For more information on the Multicomponent Plot screen, open the 7500 Software Help Information by clicking or pressing F1. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 209: View The Raw Data Plot

    The legend displays the color code for each row of the reaction plate. In the Note: example shown below, Row A is red, Row B is yellow/green, Row C is green, and so on. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 210 Guidelines filter: • Characteristic signal growth • No abrupt changes or dips For More For more information on the Raw Data Plot screen, open the 7500 Software Help by Information clicking or pressing F1. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve...

  • Page 211: Section 9.3: Perform A Study Of Multiple Experiments

    Publish the Data ..........202 Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C...

  • Page 212: Section Overview

    • In the second half, you view results in a example study file that is installed with the 7500 software. You can find the data file for the example study on your computer at: <drive>:\Applied Biosystems\<software name>\experiments\ Comparative Ct Study Example.edm...

  • Page 213: Design A Study

    Chapter 9 Analyze the Comparative C Experiment Design a Study Design a Study This section explains how to use the 7500 software to set up a comparative C example study. It also describes Applied Biosystems recommended best practices as you create the study...
  • Page 214 7. In the Set Up Experiments table, select the Comparative Ct Example experiment. The 7500 software displays the details of the experiment in the Properties pane. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 215 • Filter the experiments added to the study to simplify the list for easier review. See “How to Simplify Data Lists Using the Filter Query” on page 192. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 216 Click the Comments field, then enter Example Biological Replicate Group for the Example Comparative Ct Study. In the Select Plate drop-down list, select Comparative Ct Experiment.eds. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 217 The 7500 software automatically adds technical replicates of the selected wells to the biological group. Click OK. 4. Select the LKBGP group that you just added to the study. The 7500 software displays the details of the biological group in the Properties pane. Notes...
  • Page 218 To change the name of the group, click the Edit button after the Biological Note: Group Name field. 3. To save your changes, click OK. To leave the group unchanged, click Cancel. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 219: Analyze The Study

    Experiment Analyze the Study Analyze the Study This section explains how to use the 7500 software to analyze the comparative C example study. It also describes Applied Biosystems recommended best practices as you perform the analysis. The Comparative Ct Study Example.edm file is not designed to demonstrate the Note: use of biological replicate groups;...
  • Page 220 A hyphen (–) indicates that one or more members of the group have been omitted. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 221 1. When the result is displayed in the Log10 graph type, the expression level of the reference sample appears as 0 in the graph (log10 of 1 = 0). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 222 The maximum includes the variability associated with the endogenous control and targets in only the test samples. ▲ Flag The number of QC flags the well generated as listed in the symbol. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 223 Table” on page 162, or open the 7500 Software Help by clicking or pressing F1. • Calculating Relative Quantitation Values – Refer to the User Bulletin #2: Relative Quantitation of Gene Expression. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 224 The procedure for omitting wells is identical to that for the Amplification Plot. See “Omit Wells using the Well Table” above. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 225 Amplification Plot. See “Omit Wells using the Well Table” on page 200. View the QC Summary The QC Summary screen displays a list of the 7500 software flags, and it includes the flag frequency and location for the experiments added to the open study. The summary is identical to the QC Summary that appears in the experiment-level analysis.

  • Page 226: Publish The Data

    You can publish the study data in the same way you publish experiment data. See “Publish the Data” on page 97 for more information. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 227: Appendix A Alternate Experiment Workflows

    Help from within Applied Biosystems 7500/7500 Fast Real-Time PCR Software v2.0 by pressing F1, clicking in the toolbar, or selecting Help 7500 Software Help. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 228: Advanced Setup Workflow

    Appendix A Alternate Experiment Workflows Advanced Setup Workflow Advanced Setup Workflow When you create an experiment using Advanced Setup in the 7500 software, you can set up the experiment according to your own design. 1. Double-click (7500 software) or select Start All Programs Applied Biosystems 7500 Software <software name>...
  • Page 229 In the navigation pane, select an analysis screen to view the data (for example, select QC Summary to view a quality summary of the data). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 230: Quickstart Workflow

    Load the reaction plate into the instrument. Start the run. (Optional) Monitor the run. Unload the reaction plate from the instrument. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 231 In the navigation pane, select an analysis screen to view the data (for example, select QC Summary to view a quality summary of the data). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 232: Template Workflow

    3. (Optional) If you want to modify the experiment, use Advanced Setup (see page 204). 4. Click Save, enter a file name, then click Save to save the experiment. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 233 In the navigation pane, select an analysis screen to view the data (for example, select QC Summary to view a quality summary of the data). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 234: Export/Import Workflow

    Replace the parameters of the text file as needed. When you finish, save the file as a tab-delimited text file. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 235 Load the reaction plate into the instrument. Start the run. (Optional) Monitor the run. Unload the reaction plate from the instrument. Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 236 In the navigation pane, select an analysis screen to view the data (for example, select QC Summary to view a quality summary of the data). Notes Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 237: Bibliography

    Saiki, R.K., Scharf, S., Faloona, F., et al. 1985. Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 238 Bibliography Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 239: Glossary

    Glossary Advanced Setup In the 7500 software, a feature that allows you to set up your experiment according to your experiment design. Advanced Setup provides you with maximum flexibility in the design and setup of your experiment. See assay information file (AIF).
  • Page 240 See also cycling stage. assay In the 7500/7500 Fast system, a PCR reaction mix that contains primers to amplify a target and a reagent to detect the amplified target. ®...
  • Page 241 In presence/absence experiments, a reaction that contains IPC blocking agent, which blocks amplification of the internal positive control (IPC). In the 7500 software, the task for the IPC target in wells that contain IPC blocking agent. See also negative control-blocked IPC wells.
  • Page 242 The instrument transforms the signal to electronic data, and the data are saved in the experiment file. In the 7500 software, a data collection point is indicated by an icon in the thermal profile: •...
  • Page 243 In presence/absence experiments, abbreviation for internal positive control (IPC). In the 7500 software, the task for the IPC target in wells that contain the IPC and do not contain IPC blocking agent. See also internal positive control (IPC).
  • Page 244 PCR run. negative control In the 7500 software, the task for targets or SNP assays in wells that contain water or buffer (NC) instead of sample. No amplification of the target should occur in negative control wells.
  • Page 245 7500/7500 Fast system, the grid contains 8 rows and 12 columns. In the 7500 software, you can use the plate layout as a selection tool to assign well contents, to view well assignments, and to view results. The plate layout can be printed, included in a report, exported, and saved as a slide for a presentation.
  • Page 246 Glossary QuickStart A feature in 7500/7500 Fast systems that allows you to run an experiment without entering plate setup information. value Regression coefficient calculated from the regression line in the standard curve. The R value indicates the closeness of fit between the standard curve regression line and the individual C data points from the standard reactions.
  • Page 247 ROX dye is used as the passive reference. rs number See refSNP ID. run method Definition of the reaction volume and the thermal profile for the 7500/7500 Fast instrument run. sample The template that you are testing. Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve...
  • Page 248 SNP Assay Library In the 7500 software, a collection of SNP assays to add to genotyping experiments. The SNP assays in the library contain the SNP assay name, SNP assay color, and for each allele, the allele name or base(s), reporter, quencher, and allele colors.
  • Page 249 For example, the standard stock is used to prepare the first dilution point, the first dilution point is used to prepare the second dilution point, and so on. In the 7500 software, the volumes needed to prepare a standard dilution series are calculated by the number of dilution points, the number of standard replicates, the starting quantity, the serial factor, and the standard concentration in the stock.
  • Page 250 The nucleic acid sequence that you want to amplify and detect. target color In the 7500 software, a color assigned to a target to identify the target in the plate layout and analysis plots. Target Library In the 7500 software, a collection of targets to add to experiments. The targets in the library contain the target name, reporter, quencher, and target color.
  • Page 251 See melting temperature (Tm). unknown In the 7500 software, the task for the target or SNP assay in wells that contain the sample you are testing: • In quantitation experiments, the task for the target in wells that contain a sample with unknown target quantities.
  • Page 252 Glossary Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...

  • Page 253: Index

    105 chemical waste safety xix publish the data 174 column view Amplification Plot screen 85 Mean 95 view analysis settings 100 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 254 188 ergonomics, safety xxi publish 174 example experiment, run 66 ∆C Mean column 164 Experiment Properties screen 22 ∆C SD column 164 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 255 51 workflow 50 prepare for run 67 Methods & Materials screen 24 print reaction setup instructions 41 monitor run publish data 174 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 256 62 Run Method library 36 Standards screen 29 Run Method screen 35 study monitor during a run 73 about 188 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 257 12 with your own experiments 13 WARNING, description xii waste disposal, guidelines xix Well Table 93 wells negative control 32 Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 258 Index Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve and Comparative C Experiments...
  • Page 260 Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our Web site at www.appliedbiosystems.com.

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