abi 7500 User manual

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

Applied Biosystems
7500/7500 Fast
Real-Time PCR System
Relative Standard Curve and
Comparative CTExperiments
Get Started
Design the
Relative Standard
Curve Experiment
Prepare the
Relative Standard
Curve Reactions
Run the Relative
Standard Curve
Experiment
Analyze the
Relative Standard
Curve Experiment
Design the
Comparative CT
Experiment
Prepare the
Comparative CT
Reactions
Run the
Comparative CT
Experiment
Analyze the
Comparative CT
Experiment
Getting Started Guide

© Copyright 2007, 2010 Applied Biosystems. All rights reserved.
Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document.
APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT
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BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCID-
ENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,
INCLUDING BUT NOT LIMITED TO THE USE THEREOF.
For Research Use Only. Not for use in diagnostic procedures.
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. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln
Centre Drive, Foster City, California 94404, USA.
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of Appl ied Biosystems or its subsidiaries in the U.S. and/or certain other countries.
AmpErase, AmpliTaq Gold, and TaqMan are registered trademarks of Roche Molecular Systems, Inc.
Ambion, RNA later , RNAqueous, RNase Zap , and Silencer are registered trademarks, and DNA Zap , Pre-miR, RiboPure, and TURBO DNA- free are trademarks of
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All other trademarks are the sole property of their respective owners.
Part Number 4387783 Rev. C
06/2010

Contents
iii
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
Safety Conventions Used in This Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii
Symbols on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
Safety Labels on Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
General Instrument Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi
Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii
Chemical Waste Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix
Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xx
Physical Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xx
Biological Hazard Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi
Workstation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi
Safety and Electromagnetic Compatibility (EMC) Standards . . . . . . . . . . . . . . . . . . . xxii
Chapter 1 Get Started. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
About the 7500/7500 Fast System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Supported Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
About Relative Standard Curve and Comparative CTExperiments . . . . . . . . . . . . . . . . 7
How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
About the Example Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Example Experiment Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Chapter 2 Design the Relative Standard Curve Experiment. . . . 19
Chapter Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Create a New Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Define the Experiment Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Define the Methods and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Set Up the Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Set Up the Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Set Up the Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Set Up the Relative Quantitation Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Set Up the Run Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Review the Reaction Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
iv
Order Materials for the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Finish the Design Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Chapter 3 Prepare the Relative Standard Curve Reactions. . . . . 49
Chapter Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Prepare the Template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Prepare the Sample Dilutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Prepare the Standard Dilution Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Prepare the Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Prepare the Reaction Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Chapter 4 Run the Relative Standard Curve Experiment. . . . . . . 65
Chapter Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Prepare for the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Enable the Notification Settings (Optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Start the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Monitor the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Unload the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Chapter 5 Analyze the Relative Standard Curve Experiment . . . 75
Chapter Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Section 5.1: Review Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Analyze the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
View the Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
View the Amplification Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
View the Gene Expression Plot and Well Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Publish the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Section 5.2: Troubleshoot (If Needed) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
View the Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
View the QC Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Omit Wells from the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
View the Multicomponent Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
View the Raw Data Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Chapter 6 Design the Comparative CTExperiment . . . . . . . . . . 111
Chapter Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Create a New Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Define the Experiment Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

v
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
Define the Methods and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Set Up the Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Set Up the Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Set Up the Relative Quantitation Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
Set Up the Run Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Review the Reaction Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Order Materials for the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Finish the Design Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Chapter 7 Prepare the Comparative CT Reactions . . . . . . . . . . 137
Chapter Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Prepare the Template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Prepare the Sample Dilutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Prepare the Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
Prepare the Reaction Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Chapter 8 Run the Comparative CTExperiment . . . . . . . . . . . . 151
Chapter Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Prepare for the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Run the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Chapter 9 Analyze the Comparative CT Experiment . . . . . . . . . 155
Chapter Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Section 9.1: Review Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Analyze the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
View the Gene Expression Plot and Well Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
View the Amplification Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Publish the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
Section 9.2: Troubleshoot (If Needed) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
View the Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
View the QC Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Omit Wells from the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
View the Multicomponent Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
View the Raw Data Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Section 9.3: Perform a Study of Multiple Experiments . . . . . . . . . . . . . . . . . . . . . . 187
Section Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
Design a Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Analyze the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Publish the Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
vi
Appendix A Alternate Experiment Workflows . . . . . . . . . . . . . . . . . . 203
Advanced Setup Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
QuickStart Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Template Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
Export/Import Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

vii
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
Preface
How to Use This Guide
About the System
Documentation
The guides listed below are shipped with the Applied Biosystems 7500 and 7500 Fast
Real-Time PCR Systems.
Guide Purpose and Audience PN
Applied Biosystems 7500/7500 Fast Real-
Time PCR System Getting Started Guide
for Genotyping Experiments
Explains how to perform experiments on the 7500/7500 Fast
system. Each Getting Started Guide functions as both a:
• Tutorial, using example experiment data provided with the
Applied Biosystems 7500/7500 Fast Real-Time PCR
Software (7500/7500 Fast software).
• Guide for your own experiments.
Intended for laboratory staff and principal investigators who
perform experiments using the 7500/7500 Fast system.
4387784
Applied Biosystems 7500/7500 Fast Real-
Time PCR System Getting Started Guide
for Presence/Absence Experiments
4387785
Applied Biosystems 7500/7500 Fast Real-
Time PCR System Getting Started Guide
for Relative Standard Curve and
Comparative CTExperiments
4387783
Applied Biosystems 7500/7500 Fast Real-
Time PCR System Getting Started Guide
for Standard Curve Experiments
4387779
Applied Biosystems 7500/7500 Fast Real-
Time PCR System Maintenance Guide
Explains how to install and maintain the 7500/7500 Fast system.
Intended for laboratory staff responsible for the installation and
maintenance of the 7500/7500 Fast system.
4387777
Applied Biosystems 7500/7500 Fast Real-
Time PCR System Installation Quick
Reference Card
4387778
Real Time PCR System Reagent Guide Provides information about the reagents you can use on the
7500/7500 Fast system, including:
• An introduction to TaqMan®and SYBR®Green reagents.
• Descriptions and design guidelines for the following
experiment types:
– Quantitation experiments
– Genotyping experiments
– Presence/absence experiments
Intended for laboratory staff and principal investigators who
perform experiments using the 7500/7500 Fast system.
4387787
Applied Biosystems 7500/7500 Fast Real-
Time PCR System Site Preparation Guide
Explains how to prepare your site to receive and install the
7500/7500 Fast system.
Intended for personnel who schedule, manage, and perform the
tasks required to prepare your site for installation of the
7500/7500 Fast system.
4387776

Preface
How to Use This Guide
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
viii
Assumptions This guide assumes that you:
• Are familiar with the Microsoft Windows®XP operating system.
• Are familiar with the Internet and Internet browsers.
• 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.
Text Conventions This guide uses the following conventions:
•Bold text indicates user action. For example:
Enter 0, then press Enter for each of the remaining fields.
• Italic text indicates new or important words and is also used for emphasis. For example:
Before analyzing, always prepare fresh matrix.
• A right arrow symbol () separates successive commands you select from a drop-
down list or shortcut menu. For example:
Select FileOpen.
User Attention
Words
Two user attention words appear in Applied Biosystems user documentation. Each word
implies a particular level of observation or action as described below:
Note: – Provides information that may be of interest or help but is not critical to the use
of the product.
IMPORTANT! – Provides information that is necessary for proper instrument operation,
accurate reagent kit use, or safe use of a chemical.
Examples of the user attention words appear below:
Note: The Calibrate function is also available in the Control Console.
IMPORTANT! To verify your client connection, you need a valid user ID.
Applied Biosystems 7500/7500 Fast Real-
Time PCR Software v2.0 Help
Explains how to use the 7500/7500 Fast software to:
• Set up, run, and analyze experiments using the 7500/7500
Fast system.
• Calibrate a 7500/7500 Fast instrument.
• Verify the performance of a 7500/7500 Fast instrument with
an RNase P run.
Intended for:
• Laboratory staff and principal investigators who perform
experiments using the 7500/7500 Fast system.
• Laboratory staff responsible for the installation and
maintenance of the 7500/7500 Fast system.
NA
Guide Purpose and Audience PN

Preface
How to Obtain More Information
ix
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
Safety Alert
Words
Safety alert words also appear in user documentation. For more information, see “Safety
Alert Words” on page xii.
How to Obtain More Information
Related
Documentation
Documents Related to Genotyping Experiments
Documents Related to Presence/Absence Experiments
Documents Related to Relative Standard Curve and Comparative CTExperiments
Document PN
Allelic Discrimination Pre-Developed TaqMan®Assay Reagents Quick Reference
Card
4312212
Custom TaqMan®Genomic Assays Protocol Submission Guidelines 4367671
Custom TaqMan®SNP Genotyping Assays Protocol 4334431
Ordering TaqMan®SNP Genotyping Assays Quick Reference Card 4374204
Performing a Custom TaqMan®SNP Genotyping Assay for 96-Well Plates Quick
Reference Card
4371394
Performing a TaqMan®Drug Metabolism Genotyping Assay for 96-Well Plates
Quick Reference Card
4367636
Pre-Developed TaqMan®Assay Reagents Allelic Discrimination Protocol 4312214
TaqMa n®Drug Metabolism Genotyping Assays Protocol 4362038
TaqMa n®SNP Genotyping Assays Protocol 4332856
Document PN
DNA Isolation from Fresh and Frozen Blood, Tissue Culture Cells, and Buccal
Swabs Protocol
4343586
NucPrep®Chemistry: Isolation of Genomic DNA from Animal and Plant Tissue
Protocol
4333959
Document PN
Amplification Efficiency of TaqMan®Gene Expression Assays Application Note 127AP05
Applied Biosystems High-Capacity cDNA Reverse Transcription Kits Protocol 4375575
Custom TaqMan®Gene Expression Assays Protocol 4334429
Primer Express®Software Version 3.0 Getting Started Guide 4362460
TaqMa n®Gene Expression Assays Protocol 4333458
User Bulletin #2: Relative Quantitation of Gene Expression 4303859

Preface
How to Obtain More Information
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
x
Documents Related to Standard Curve Experiments
Documents Related to the Reagent Guide
Note: For more documentation, see “How to Obtain Support” on page xi.
Document PN
Amplification Efficiency of TaqMan®Gene Expression Assays Application Note 127AP05
Custom TaqMan®Gene Expression Assays Protocol 4334429
Primer Express®Software Version 3.0 Getting Started Guide 4362460
TaqMa n®Gene Expression Assays Protocol 4333458
User Bulletin #2: Relative Quantitation of Gene Expression 4303859
Document PN
Applied Biosystems High-Capacity cDNA Reverse Transcription Kits Protocol 4375575
Custom TaqMan®Gene Expression Assays Protocol 4334429
Custom TaqMan®Genomic Assays Protocol: Submission Guidelines 4367671
Custom TaqMan®SNP Genotyping Assays Protocol 4334431
Power SYBR®Green PCR Master Mix and RT-PCR Protocol 4367218
Pre-Developed TaqMan®Assay Reagents Allelic Discrimination Protocol 4312214
Primer Express®Software Version 3.0 Getting Started Guide 4362460
SYBR®Green PCR and RT-PCR Reagents Protocol 4304965
SYBR®Green PCR Master Mix and RT-PCR Reagents Protocol 4310251
TaqMa n®Drug Metabolism Genotyping Assays Protocol 4362038
TaqMa n®Exogenous Internal Positive Control Reagents Protocol 4308335
TaqMa n®Fast Universal PCR Master Mix (2 ✕ ) Protocol 4351891
TaqMa n®Gene Expression Assays Protocol 4333458
TaqMa n®Gene Expression Master Mix Protocol 4371135
TaqMa n®Genotyping Master Mix Protocol 4371131
TaqMa n®SNP Genotyping Assays Protocol 4332856
TaqMa n®Universal PCR Master Mix Protocol 4304449
User Bulletin #2: Relative Quantitation of Gene Expression 4303859
Using TaqMan®Endogenous Control Assays to Select an Endogenous Control
for Experimental Studies Application Note
127AP08

Preface
How to Obtain Support
xi
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
Obtaining
Information from
the Software Help
The 7500 Software Help describes how to use each feature of the touchscreen. Open the
Help from within the software by doing one of the following:
• Press F1.
• Click in the toolbar.
• Select Help7500 Software Help.
To find topics of interest in the Help:
• Review the table of contents.
• Search for a specific topic.
• Search the alphabetized index.
Send Us Your
Comments
Applied Biosystems welcomes your comments and suggestions for improving its user
documents. You can e-mail your comments to:
[email protected]
IMPORTANT! The e-mail address above is only for submitting comments and
suggestions relating to documentation. To order documents, download PDF files, or for
help with a technical question, go to http://www.appliedbiosystems.com, then click the
link for Support. (See “How to Obtain Support” on page xi).
How to Obtain Support
For the latest services and support information for all locations, go to
http://www.appliedbiosystems.com, then click the link for Support.
At the Support page, you can:
• Search through frequently asked questions (FAQs).
• Submit a question directly to Technical Support.
• Order Applied Biosystems user documents, MSDSs, certificates of analysis, and
other related documents.
• Download PDF documents.
• Obtain information about customer training.
• Download software updates and patches.
In addition, the Support page provides access to worldwide telephone and fax numbers
to contact Applied Biosystems Technical Support and Sales facilities.
IMPORTANT! When directed to do so by this guide, or when you need to schedule
maintenance for your 7500/7500 Fast instrument (such as annual planned maintenance
or temperature verification/calibration), contact the Applied Biosystems Care Center. To
obtain a phone number for or to send an e-mail to the center, go to
http://www.appliedbiosystems.com/support/contact.

Preface
Safety Conventions Used in This Document
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
xii
Safety Conventions Used in This Document
Safety Alert
Words
Four safety alert words appear in Applied Biosystems user documentation at points in
the document where you need to be aware of relevant hazards. Each alert
word–IMPORTANT, CAUTION, WARNING, DANGER–implies a particular level of
observation or action, as defined below.
Definitions
IMPORTANT! – Indicates information that is necessary for proper instrument operation,
accurate chemistry kit use, or safe use of a chemical.
– Indicates a potentially hazardous situation that, if not avoided, may
result in minor or moderate injury. It may also be used to alert against unsafe practices.
– Indicates a potentially hazardous situation that, if not avoided,
could result in death or serious injury.
– Indicates an imminently hazardous situation that, if not avoided,
will result in death or serious injury. This signal word is to be limited to the most extreme
situations.
Except for IMPORTANTs, each safety alert word in an Applied Biosystems document
appears with an open triangle figure that contains a hazard symbol. These hazard
symbols are identical to the hazard icons that are affixed to Applied Biosystems
instruments (see “Safety Symbols” on page xiii).
Examples
The following examples show the use of safety alert words:
IMPORTANT! Wear powder-free gloves when you handle the halogen lamp.
The lamp is extremely hot. Do not touch the lamp until it has cooled to
room temperature.
CHEMICAL HAZARD. Ethanol is a flammable liquid and vapor.
Exposure causes eye, skin, and respiratory tract irritation and may cause central nervous
system depression and liver damage. Read the MSDS, and follow the handling instructions.
Wear appropriate protective eyewear, clothing, and gloves.
ELECTRICAL HAZARD. Failure to ground the instrument properly
can lead to an electrical shock. Ground the instrument according to the provided instructions.

Preface
Symbols on Instruments
xiii
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
Symbols on Instruments
Electrical
Symbols on
Instruments
The following table describes the electrical symbols that may be displayed on
Applied Biosystems instruments.
Safety Symbols The following table describes the safety symbols that may be displayed on
Applied Biosystems instruments. Each symbol may appear by itself or in combination
with text that explains the relevant hazard (see “Safety Labels on Instruments” on
page xiv). These safety symbols may also appear next to DANGERS, WARNINGS, and
CAUTIONS that occur in the text of this and other product-support documents.
Symbol Description Symbol Description
Indicates the On position of the
main power switch.
Indicates a terminal that may be
connected to the signal ground
reference of another instrument.
This is not a protected ground
terminal.
Indicates the Off position of the
main power switch.
Indicates a protective grounding
terminal that must be connected
to earth ground before any other
electrical connections are made
to the instrument.
Indicates a standby switch by
which the instrument is switched
on to the Standby condition.
Hazardous voltage may be
present if this switch is on
standby.
Indicates a terminal that can
receive or supply alternating
current or voltage.
Indicates the On/Off position of a
push-push main power switch.
Indicates a terminal that can
receive or supply alternating or
direct current or voltage.
Symbol Description
Indicates that you should consult the manual for further information and to
proceed with appropriate caution.
Indicates the presence of an electrical shock hazard and to proceed with
appropriate caution.
Indicates the presence of a hot surface or other high-temperature hazard and
to proceed with appropriate caution.
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.

Preface
Safety Labels on Instruments
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
xiv
Environmental
Symbols on
Instruments
The following symbol applies to all Applied Biosystems electrical and electronic
products placed on the European market after August 13, 2005.
Safety Labels on Instruments
The following CAUTION, WARNING, and DANGER statements may be displayed on
Applied Biosystems instruments in combination with the safety symbols described in the
preceding section.
Symbol Description
Do not dispose of this product as unsorted municipal waste. Follow local
municipal waste ordinances for proper disposal provisions to reduce the
environmental impact of waste electrical and electronic equipment (WEEE).
European Union customers:
Call your local Applied Biosystems Customer Service office for equipment
pick-up and recycling. See http://www.appliedbiosystems.com for a list of
customer service offices in the European Union.
English Français
CAUTION Hazardous chemicals. Read the
Material Safety Data Sheets (MSDSs) before
handling.
ATTENTION Produits chimiques dangeureux.
Lire les fiches techniques de sûreté de
matériels avant la manipulation des produits.
CAUTION Hazardous waste. Refer to
MSDS(s) and local regulations for handling
and disposal.
ATTENTION Déchets dangereux. Lire les
fiches techniques de sûreté de matériels et la
régulation locale associées à la manipulation
et l'élimination des déchets.
WARNING Hot lamp. AVERTISSEMENT Lampe brûlante.
WARNING Hot. Replace lamp with an
Applied Biosystems lamp.
AVERTISSEMENT Composants brûlants.
Remplacer la lampe par une lampe
Applied Biosystems.
CAUTION Hot surface. ATTENTION Surface brûlante.
DANGER High voltage. DANGER Haute tension.
WARNING To reduce the chance of electrical
shock, do not remove covers that require tool
access. No user-serviceable parts are inside.
Refer servicing to Applied Biosystems
qualified service personnel.
AVERTISSEMENT Pour éviter les risques
d'électrocution, ne pas retirer les capots dont
l'ouverture nécessite l'utilisation d'outils.
L’instrument ne contient aucune pièce
réparable par l’utilisateur. Toute intervention
doit être effectuée par le personnel de service
qualifié de Applied Biosystems.
CAUTION Moving parts. ATTENTION Parties mobiles.
WARNING This instrument is designed for
12 V, 75 W Halogen lamps only.
AVERTISSEMENT Cet instrument est conçu
pour des lampes d'halogène de 12 V et 75 W
seulement.

Preface
Safety Labels on Instruments
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Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
Locations of
Warnings
The Applied Biosystems 7500/7500 Fast Real-Time PCR System contains warnings at
the locations shown below.
Attention
Physical hazard
Physical hazard
Attention
Attention
Attention

Preface
General Instrument Safety
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
xvi
General Instrument Safety
PHYSICAL INJURY HAZARD. Use this product only as
specified in this document. Using this instrument in a manner not specified by
Applied Biosystems may result in personal injury or damage to the instrument.
Moving and
Lifting the
Instrument
PHYSICAL INJURY HAZARD. The instrument is to be moved
and positioned only by the personnel or vendor specified in the applicable site
preparation guide. If you decide to lift or move the instrument after it has been installed,
do not attempt to lift or move the instrument without the assistance of others, the use of
appropriate moving equipment, and proper lifting techniques. Improper lifting can cause
painful and permanent back injury. Depending on the weight, moving or lifting an
instrument may require two or more persons.
Moving and
Lifting Stand-
Alone Computers
and Monitors
Do not attempt to lift or move the computer or the monitor without
the assistance of others. Depending on the weight of the computer and/or the monitor,
moving them may require two or more people.
Things to consider before lifting the computer and/or the monitor:
• Make sure that you have a secure, comfortable grip on the computer or the monitor
when lifting.
• Make sure that the path from where the object is to where it is being moved is clear
of obstructions.
• Do not lift an object and twist your torso at the same time.
• Keep your spine in a good neutral position while lifting with your legs.
• Participants should coordinate lift and move intentions with each other before
lifting and carrying.
• Instead of lifting the object from the packing box, carefully tilt the box on its side
and hold it stationary while someone slides the contents out of the box.
Operating the
Instrument
Ensure that anyone who operates the instrument has:
• Received instructions in both general safety practices for laboratories and specific
safety practices for the instrument.
• Read and understood all applicable Material Safety Data Sheets (MSDSs). See
“About MSDSs” on page xvii.
PHYSICAL INJURY HAZARD. Use this instrument as specified
by Applied Biosystems. Using this instrument in a manner not specified by
Applied Biosystems may result in personal injury or damage to the instrument.
Cleaning or
Decontaminating
the Instrument
Before using a cleaning or decontamination method other than those
recommended by the manufacturer, verify with the manufacturer that the proposed
method will not damage the equipment.

Preface
Chemical Safety
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Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
Chemical Safety
Chemical Hazard
Warning
CHEMICAL HAZARD. Before handling any chemicals, refer to
the Material Safety Data Sheet (MSDS) provided by the manufacturer, and observe all
relevant precautions.
CHEMICAL HAZARD. All chemicals in the instrument, including
liquid in the lines, are potentially hazardous. Always determine what chemicals have
been used in the instrument before changing reagents or instrument components. Wear
appropriate eyewear, protective clothing, and gloves when working on the instrument.
CHEMICAL STORAGE HAZARD. Never collect or store waste
in a glass container because of the risk of breaking or shattering. Reagent and waste
bottles can crack and leak. Each waste bottle should be secured in a low-density
polyethylene safety container with the cover fastened and the handles locked in the
upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent
and waste bottles.
About MSDSs Chemical manufacturers supply current Material Safety Data Sheets (MSDSs) with
shipments of hazardous chemicals to new customers. They also provide MSDSs with the
first shipment of a hazardous chemical to a customer after an MSDS has been updated.
MSDSs provide the safety information you need to store, handle, transport, and dispose
of the chemicals safely.
Each time you receive a new MSDS packaged with a hazardous chemical, be sure to
replace the appropriate MSDS in your files.
Obtaining MSDSs The MSDS for any chemical supplied by Applied Biosystems is available to you free
24 hours a day. To obtain MSDSs:
1. Go to www.appliedbiosystems.com, click Support, then click MSDS Search.
2. In the Keyword Search field, enter the chemical name, product name, MSDS part
number, or other information that appears in the MSDS of interest. Select the
language of your choice, then click Search.
3. Find the document of interest, right-click the document title, then select any of the
following:
•Open – To view the document
•Print Target – To print the document
•Save Target As – To download a PDF version of the document to a destination
that you choose
Note: For the MSDSs of chemicals not distributed by Applied Biosystems, contact the
chemical manufacturer.

Preface
Chemical Safety
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Relative Standard Curve
and Comparative CTExperiments
xviii
Chemical Safety
Guidelines
To minimize the hazards of chemicals:
• Read and understand the Material Safety Data Sheets (MSDSs) provided by the
chemical manufacturer before you store, handle, or work with any chemicals or
hazardous materials. (See “About MSDSs” on page xvii.)
• Minimize contact with chemicals. Wear appropriate personal protective equipment
when handling chemicals (for example, safety glasses, gloves, or protective
clothing). For additional safety guidelines, consult the MSDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use
only with adequate ventilation (for example, fume hood). For additional safety
guidelines, consult the MSDS.
• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the
manufacturer’s cleanup procedures as recommended on the MSDS.
• Comply with all local, state/provincial, or national laws and regulations related to
chemical storage, handling, and disposal.