Applied Biosystems
7300, 7500 and 7500 Fast Absolute Quantification Getting Started Guide Rev B June 2010
Getting Started Guide
96 Pages

Preview
Page 1
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System
Introduction and Example AQ Experiment
Absolute Quantification Getting Started Guide
Designing an AQ Experiment
Primer Extended on mRNA 5′
3′ Reverse Primer
5′ cDNA
Oligo d(T) or random hexamer
Synthesis of 1st cDNA strand 5′ cDNA
3′
Performing Reverse Transcription
STANDARD
Generating Data from AQ Plates – 7300 or Standard 7500 System
FAST
Generating Data from AQ Plates – 7500 Fast System
Analyzing Data in an AQ Study
© Copyright 2006, 2010 Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. NOTICE TO PURCHASER: The Applied Biosystems 7300, 7500 and 7500 Fast Real-Time PCR Systems are real-time thermal cyclers covered by US patents and corresponding claims in their non-US counterparts, owned by Applied 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. Trademarks Applera, Applied Biosystems, AB (Design), ABI PRISM, MicroAmp, Primer Express, and VIC are registered trademarks and FAM, MultiScribe, ROX, and TAMRA are trademarks of Applied Biosystems or its subsidiaries in the US and/or certain other countries. AmpErase, AmpliTaq Gold, and TaqMan are registered trademarks of Roche Molecular Systems, Inc. SYBR is a registered trademark of Molecular Probes, Inc. Microsoft and Windows are registered trademarks of Microsoft Corporation. All other trademarks are the sole property of their respective owners. Part Number 4378656 Rev. B 06/2010
Absolute Quantification Experiment Workflow
Chapter 1
Introduction
About the 7300/7500/7500 Fast system
About absolute quantification
About AQ experiments
Chapter 2
Designing an AQ Experiment
Overview
Specify components of an AQ experiment
Select the chemistry
Performing Reverse Transcription
Isolate total RNA
Adjust RNA concentration
Convert total RNA to cDNA
Chapter 3
Chapter 4
Primer Extended on mRNA 5´
3´ Reverse Primer
5´ cDNA
Oligo d(T) or random hexamer
Synthesis of 1st cDNA strand 5´ cDNA
3´
STANDARD
Chapter 6
FAST
Choose probes and primers
Primer Extended on mRNA 5´ 5´ cDNA
5´ cDNA
STANDARD
Prepare the PCR Master Mix
Prepare the reaction plate
Create a new AQ Plate document
Program the thermal cycling conditions
Save the AQ Plate document
Start the run
Running an AQ Plate Fast
Analyzing AQ Data
3´ Reverse Primer
Oligo d(T) or random hexamer
Synthesis of 1st cDNA strand 3´
Running an AQ Plate Standard
(OR)
Chapter 5
Select one-step or two-step RT-PCR
FAST
Configure analysis settings
Adjust the baseline and threshold
Absolute Quantification Getting Started Guide for the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System
Analyze and view results
If necessary, omit samples
Export the AQ Plate document, if desired
iii
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Absolute Quantification Getting Started Guide for the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System
Contents
Absolute Quantification Experiment Workflow
iii
Preface
vii
How to Use This Guide... vii How to Obtain More Information... ix How to Obtain Support... x
Chapter 1
Introduction
1
About the 7300/7500/7500 Fast System... 2 About Absolute Quantification... 2 About AQ Experiments... 3
Chapter 2
Designing an AQ Experiment
7
Overview... 8 Specifying the Components of an AQ Experiment... 8 Selecting the Chemistry... 9 Selecting One- or Two-Step RT-PCR... 10 Choosing the Probes and Primers... 12
Chapter 3
Performing Reverse Transcription
13
Guidelines for Preparing RNA... 14 Converting Total RNA to cDNA... 15
Chapter 4
Running an AQ Plate – 7300 or Standard 7500 System
17
Before You Begin... 18 Preparing the PCR Master Mix... 18 Preparing the Reaction Plate... 19 Creating an Absolute Quantification (AQ) Plate Document... 21 Specifying Thermal Cycling Conditions and Starting the Run... 25
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
v
Chapter 5
Running an AQ Plate – 7500 Fast System
31
Before You Begin... 32 Preparing the PCR Master Mix... 32 Preparing the Reaction Plate... 34 Creating an Absolute Quantification (AQ) Plate Document... 37 Specifying Thermal Cycling Conditions and Starting the Run... 41 Troubleshooting... 45
Chapter 6
Analyzing AQ Data
47
Configuring Analysis Settings... 48 Adjusting the Baseline and Threshold... 49 Analyzing and Viewing the AQ Data... 55 Omitting Samples... 62 Exporting AQ Plate Data... 64
Appendix A Creating Detectors
65
Appendix B Guidelines for Generating Standard Curves
67
Appendix C Dissociation-curve Analysis
69
Appendix D Example AQ Experiment
71
References
79
Index
81
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Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
Preface How to Use This Guide Purpose of This Guide Assumptions
This manual is written for principal investigators and laboratory staff who conduct absolute quantification assays using the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System. This guide assumes that you have: • Familiarity with Microsoft® Windows® XP operating system. • Knowledge of general techniques for handling DNA and RNA samples and preparing them for PCR. • A general understanding of hard drives and data storage, file transfers, and copying and pasting. • Networking experience, if you want to integrate the 7300/7500/7500 Fast system into your existing laboratory data flow system.
Text Conventions
This guide uses the following conventions: • Bold indicates user action. For example: Type 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 bracket (>) separates successive commands you select from a dropdown or shortcut menu. For example: Select File > Open > Spot Set.
User Attention Words
The following 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 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.
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
vii
Preface How to Use This Guide
Safety
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. You can obtain from Applied Biosystems the MSDS for any chemical supplied by Applied Biosystems. This service is free and available 24 hours a day. To obtain MSDSs:
1. Go to https://docs.appliedbiosystems.com/msdssearch.html 2. In the Search field, type in the chemical name, 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
4. To have a copy of a document sent by fax or e-mail, select Fax or Email to the left of the document title in the Search Results page, then click RETRIEVE DOCUMENTS at the end of the document list. After you enter the required information, click View/Deliver Selected Documents Now. Refer to the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Installation and Maintenance Getting Started Guide and the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Site Preparation Guide for important safety information.
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Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
Preface How to Obtain More Information
How to Obtain More Information Related Documentation
For more information about using the 7300/7500/7500 Fast system, refer to the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Online Help or the documents shown below. Online Help P/N
P/N
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Detection Getting Started Guide
4347821
4378652
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Allelic Discrimination Getting Started Guide
4347822
4378653
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
4347824
4378655
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Site Preparation Guide
4347823
4378654
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Installation and Maintenance Guide
4347828
4378657
Real-Time PCR Systems Chemistry Guide
4348358
4378658
Applied Biosystems 7500 FAST Real-Time PCR System, QRC
4362285
4378659
Applied Biosystems Real-Time System Computer Set Up Guide, QRC
4365367
4378660
Document Title
ABI PRISM® 7700 Sequence Detection System User Bulletin #2: Relative Quantitation of Gene Expression
Send Us Your Comments
4303859
Applied Biosystems welcomes your comments and suggestions for improving its user documents. You can e-mail your comments to: [email protected]
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
ix
Preface How to Obtain Support
How to Obtain Support To contact Applied Biosystems Technical Support from North America by telephone, call 1.800.899.5858. 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: • Obtain worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities • 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
x
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
Chapter 1
Introduction
About the 7300/7500/7500 Fast system
See page 2
About absolute quantification
See page 2
About AQ experiments
See page 3
Introduction
Designing an AQ Experiment
Primer Extended on mRNA 5´
3´ Reverse Primer
5´ cDNA
Oligo d(T) or random hexamer
Synthesis of 1st cDNA strand 3´
5´ cDNA
Performing Reverse Transcription
Running an ST T
AQ Plate R RD
OR
Standard
Analyzing AQ Data
Running an AQ Plate Fast
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
1
1
Chapter 1 Introduction About the 7300/7500/7500 Fast System
About the 7300/7500/7500 Fast System Description
The Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System uses fluorescentbased PCR chemistries to provide quantitative detection of nucleic acid sequences using real-time analysis and qualitative detection of nucleic acid sequences using end-point and dissociation-curve analysis.
Absolute Quantification Assay
The 7300/7500/7500 Fast system allows you to perform several assay types using plates in the 96-well format. This guide describes the absolute quantification (AQ) assay. For more information about the other assay types, refer to the Real-Time PCR Systems Chemistry Guide and the Online Help for the 7300/7500/7500 Fast System (Online Help).
About Absolute Quantification Definition
Absolute quantification is the process that determines the absolute quantity of a single nucleic acid target sequence within an unknown sample.
Real-time PCR Assays
AQ is performed using real-time PCR. In Real-Time PCR you monitor the progress of the PCR as it occurs. Data are collected throughout the PCR process rather than at the end of the PCR process (end-point PCR). In Real-Time PCR, reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than by the amount of target accumulated at the end of PCR.
Using AQ Plate Documents for Plus/Minus and AD Assays
Although Plus/Minus and Allelic Discrimination (AD) assays are end-point assays, Applied Biosystems recommends that you use the 7300/7500/7500 Fast system to perform amplification and view the real-time PCR results. In the event that an experiment fails, you can study the amplification plots to help determine the cause of the failure. Use AQ Plate documents to store real-time data for Plus/Minus and AD assays. AQ Plate documents for troubleshooting Plus/Minus and AD assays do not require standard curves.
Notes
2
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
Chapter 1 Introduction About AQ Experiments
About AQ Experiments 1 AQ Experiment Workflow
In this document the term “AQ experiment” refers to the entire AQ assay process beginning with generating cDNA from RNA (reverse transcription) through analyzing AQ data. The AQ experiment workflow has several steps, shown in the figure on page iii. AQ assays use a standard curve to calculate the quantity of an unknown target sequence. The results of AQ experiments are reported in the same units of measure as the standard curve. The 7300/7500/7500 Fast system store Real-Time PCR data collected from the reaction plate in an AQ Plate document. Each run consists of a single plate. The 7300/7500/7500 Fast system provide several views for analyzing data.
Plated Reactions
7300/7500/7500 Fast Instrument
SDS Software
AQ Plate Document
Analyze Results
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
3
Chapter 1 Introduction About AQ Experiments
Terms Used in Quantification Analysis
Term
Definition
Baseline
The initial cycles of PCR in which there is little change in fluorescence signal.
Threshold
A level of delta Rn-automatically determined by the SDS Software 1.3.1 or manually set-used for CT determination in real-time assays. The level is set to be above the baseline and sufficiently low to be within the exponential growth region of the amplification curve. The threshold is the line whose intersection with the Amplification plot defines the CT.
Threshold cycle (CT)
The fractional cycle number at which the fluorescence passes the threshold.
No template control (NTC)
A sample that does not contain template. It is used to verify amplification quality.
Nucleic acid target (also called “template”)
Nucleotide sequence that you want to detect or quantify.
Passive reference
A dye that provides an internal fluorescence reference to which the reporter dye signal can be normalized during data analysis. Normalization is necessary to correct for fluorescence fluctuations caused by changes in concentration or in volume.
Reporter dye
The dye attached to the 5′ end of a TaqMan® probe. The dye provides a fluorescence signal that indicates specific amplification.
Normalized reporter (Rn)
The ratio of the fluorescence emission intensity of the reporter dye to the fluorescence emission intensity of the passive reference dye.
Delta Rn (∆Rn)
The magnitude of the signal generated by the specified set of PCR conditions. (∆Rn = Rn −baseline)
Standard
A sample of known quantity used to construct a standard curve.
Unknown sample
A sample containing an unknown quantity of template that you want to characterize.
The figure below shows a representative amplification plot and includes some of the terms defined above. Rn+ Sample
Rn
Rn
Threshold
Rn– No Template Control
Baseline
0
5
10
15
GR0757
CT
20
25
30
35
40
Cycle Number
Notes
4
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
Chapter 1 Introduction About AQ Experiments
Required UserSupplied Materials
Item
Source
ABI PRISM® 6100 Nucleic Acid PrepStation
Applied Biosystems - (PN 6100-01)
High Capacity cDNA Archive Kit
Applied Biosystems - (PN 4322171)
TaqMan® Universal PCR Master Mix
Applied Biosystems - (PN 4304437)
TaqMan® Fast Universal PCR Master Mix (2X) No AmpErase® UNG
Applied Biosystems - (PN 4352042)
1
Applied Biosystems - (PN 4309169)
TaqMan® One Step RT-PCR Master Mix ABI PRISM® 96-Well Optical Reaction Plate with Barcode (code 128)
Applied Biosystems - (PN 4306737) Applied Biosystems - (PN 4346906)
Optical 96-Well Fast Thermal Cycling Plate with Barcode (code 128) Optical Adhesive Cover
Applied Biosystems - (PN 4311971)
Labeled primers and probes from one of the following sources: • TaqMan® Gene Expression Assays (predesigned primers and probes)
• Applied Biosystems Web site
• Custom TaqMan® Gene Expression Assays service (predesigned primers and probes)
• Contact your Applied Biosystems Sales Representative
• Primer Express® Software (customdesigned primers and probes) Reagent tubes with caps, 10-mL
Applied Biosystems - (PN 4305932)
Centrifuge with adapter for 96-well plates
Major laboratory supplier (MLS)
Gloves
MLS
Microcentrifuge
MLS
Microcentrifuge tubes, sterile 1.5-mL
MLS
Nuclease-free water
MLS
Pipette tips, with filter plugs
MLS
Pipettors, positive-displacement
MLS
Vortexer
MLS
Example AQ Experiment Overview
To better illustrate how to design, perform, and analyze AQ experiments, this section guides you through an example experiment. The example experiment represents a typical AQ experiment setup that you can use as a quick-start procedure to familiarize yourself with the AQ workflow. Detailed steps in the AQ workflow are described in the subsequent chapters of this guide. Included in these chapters are Example Experiment boxes that provide details for some of the related steps in the example experiment. Refer to Appendix D, “Example AQ Experiment,” on page 71 for more information.
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
5
Chapter 1 Introduction About AQ Experiments
Notes
6
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
Chapter 2
Designing an AQ Experiment
Overview
Introduction and Example AQ Experiment
Specify components of an AQ experiment
Designing an AQ Experiment
Primer Extended on mRNA 5´
3´ Reverse Primer
5´ cDNA
Oligo d(T) or random hexamer
Synthesis of 1st cDNA strand 3´
5´ cDNA
Performing Reverse Transcription
Running an ST T
AQ Plate R RD
OR
Standard
Analyzing AQ Data
Running an AQ Plate Fast
2
See page 8
See page 8
Select the chemistry
See page 9
Select one-step or two-step RT-PCR
See page 10
Choose probes and primers
See page 12
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
7
Chapter 2 Designing an AQ Experiment Overview
Overview Typical AQ experiments are designed for traditional (singleplex) PCR, where a primer pair plus a TaqMan probe or a primer pair plus a SYBR® Green binding dye are present in a reaction. The following sections describe design decisions required for AQ experiments.
Specifying the Components of an AQ Experiment For each AQ experiment, specify: • An unknown – The nucleic acid sequence that you are quantifying. • Standards – This guide assumes that you have generated a set of standards for each target sequence that you are quantifying. Appendix B on page 67 provides guidelines for generating standards. • Replicate wells – For absolute quantification assays, Applied Biosystems recommends the use of three or more replicate reactions per sample to ensure statistical significance. For more information about these requirements, refer to the Real-Time PCR Systems Chemistry Guide (PN 4378658). Sample Experiment The example experiment aims to determine the quantity of the RNase P gene in two populations. Because a single gene is being studied, only one set of standards is required (A). Four replicates of each unknown and standard are performed to ensure statistical significance. In experiments where multiple genes are being studied, a set of standards is required for each gene (B).
A. Single gene in two populations
B. Two genes in two populations
NTC NT
STD 125 1250
STD S TD 250 2500
STD 500 5000 0
STD S TD 10000 0000 000 0
STD S TD 20000 20000 2000 0
GR2364
GR2341
RNase RNa se P
Notes
8
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
Chapter 2 Designing an AQ Experiment Selecting the Chemistry
Selecting the Chemistry About Chemistries
Applied Biosystems offers two types of chemistries that you can use to detect PCR products on real-time instruments, as explained in the following table. Both TaqMan® probe-based and SYBR® Green I dye chemistries can be used for either one- or two-step RT-PCR. For more information about these chemistries, refer to the Real-Time PCR Systems Chemistry Guide.
Chemistry TaqMan® reagents or kits Description TaqMan® reagent-based chemistry uses a fluorogenic probe to enable detection of a specific PCR product as it accumulates during PCR cycles.
Process Polymerization FORWARD PRIMER
5′ 3′
3′
5′ 3′ 5′
3′ 5′
Polymerization Completed R
R Q 3′
5′ 3′
5′
5′
3′ 5′
Q 3′
5′ 3′
5′
5′
3′ 5′
Step 3: After being separated from the quencher, the reporter dye emits its characteristic fluorescence.
Step 1: Reaction setup The SYBR® Green I dye fluoresces when bound to double-stranded DNA.
Description Uses SYBR Green I dye, a double-stranded DNA binding dye, to detect PCR products as they accumulate during PCR cycles.
Step 2: Denaturation When the DNA is denatured, the SYBR® Green I dye is released and the fluorescence is drastically reduced.
Advantages • Reduces cost (no probe needed). FORWARD PRIMER
Step 3: Polymerization During extension, primers anneal and PCR product is generated.
• Yields a melting profile of distinct PCR runs.
Binds nonspecifically to all double-stranded DNA sequences. To avoid false positive signals, check for nonspecific product formation using dissociation curve or gel analysis.
5′
5′
Cleavage
SYBR® Green I reagents
Limitations
3′
Step 1 (continued): When both dyes are attached to the probe, reporter dye emission is quenched.
Step 2: During each extension cycle, the AmpliTaq Gold® DNA polymerase cleaves the reporter dye from the probe.
• Increases sensitivity for detecting amplification products relative to product length.
Q 5′ 3′
Step 1: A reporter (R) and a quencher (Q) are attached to the 5' and 3' ends of a TaqMan probe.
• Optimized assays available.
• Amplifies all double-stranded DNA.
R
Q = QUENCHER
REVERSE PRIMER
• Increases specificity with a probe. Specific hybridization between probe and target generates fluorescence signal.
• Allows 5′ -nuclease assay to be carried out during PCR.
PROBE
R = REPORTER
Q
5′
Advantages
• Provides multiplex capability.
2
Strand Displacement R
REVERSE PRIMER
Step 4: Polymerization completed SYBR® Green I dye binds to the double-stranded product, resulting in a net increase in fluorescence detected by the instrument.
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
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Chapter 2 Designing an AQ Experiment Selecting One- or Two-Step RT-PCR
Selecting One- or Two-Step RT-PCR When performing real-time PCR, you have the option of performing reverse transcription (RT) and PCR in a single reaction (one-step) or in separate reactions (twostep). The reagent configuration you use depends on whether you are performing twostep or one-step RT-PCR: • Two-step RT-PCR is performed in two separate reactions: First, total RNA is reverse transcribed into cDNA, then the cDNA is amplified by PCR. This method is useful for detecting multiple transcripts from a single cDNA template or for storing cDNA aliquots for later use. AmpErase® UNG enzyme can be used to prevent carryover contamination Users of the 7500 Fast system can use TaqMan® Fast Universal PCR Master Mix or TaqMan® Universal PCR Master Mix for an approximately 40 minute run time or a 2 hour run time, respectively. IMPORTANT! This guide assumes that AQ experiments are designed using twostep RT-PCR. For additional options, refer to the Real-Time PCR Systems Chemistry Guide.
In a two-step reaction, TaqMan® Fast Universal PCR Master Mix does not have the UNG enzyme. • In one-step RT-PCR, RT and PCR take place in one buffer system, which provides the convenience of a single-tube preparation for RT and PCR amplification. However, you cannot use the carryover prevention enzyme, AmpErase® UNG (uracil-N-glycosylase), with one-step RT-PCR. For more information about UNG, refer to the Real-Time PCR Systems Chemistry Guide.
Notes
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Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide