Applied biosystems 7500 User manual

Introduction and

Example

RQ Experiment

Designing an

RQ Experiment

Performing

Reverse

Transcription

Generating Data

from RQ Plates –

7300 or Standard

7500 System

Generating Data

from RQ Plates –

7500 Fast System

Analyzing Data in

an RQ Study

Primer Extended on mRNA

5′3′

Reverse

Primer

5′

Synthesis of 1st cDNA strand

cDNA

Oligo d(T) or random hexamer

3′5′

cDNA

STANDARD

FAST

Applied Biosystems 7300/7500/7500 Fast

Real-Time PCR System

Relative Quantification

Getting Started Guide

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

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Microsoft and Windows are registered trademarks of Microsoft Corporation.

All other trademarks are the sole property of their respective owners.

Part Number 4378655 Rev. B

06/2010

Chapter 2 Select the

PCR method

Specify the

components of

an RQ experiment

Select the

chemistry

Select one-step or

two-step RT-PCR

Choose probes

and primers

Chapter 3 Isolate total RNA Adjust RNA

concentration

Convert

total RNA to cDNA

Primer Extended on mRNA

5´ 3´

Reverse

Primer

5´

Synthesis of 1st cDNA strand

cDNA

Oligo d(T) or random hexamer

3´ 5´

cDNA

Create a new

RQ Study document

Configure

analysis settings

Adjust the

baseline and

threshold

Analyze and

view results

If necessary,

omit samples

Export AQ

Plate document,

if desired

Chapter 6

Chapter 5

Chapter 1

About the

7300/7500/7500 Fast

system

About Relative

Quantification

About

RQ Experiments

Prepare the

PCR Master Mix

Prepare the

reaction plate

Create a new

RQ Plate document

Program the

thermal cycling

conditions

Save the

RQ Plate

document

Start the run View

RQ plate data

Chapter 4

(OR)

Designing

an RQ

Experiment

Performing

Reverse

Transcription

Primer Extended on mRNA

5´ 3´

Reverse

Primer

5´

Synthesis of 1st cDNA strand

cDNA

Oligo d(T) or random hexamer

3´ 5´

cDNA

Performing

an RQ Study

Introduction

Generating

Data from

RQ Plates

Standard

Generating

Data from

RQ Plates

Fast

STANDARD

FAST

STANDARD

FAST

Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide iii

Relative Quantification Experiment Workflow

iv Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide

Contents

Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide v

Relative 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 Relative Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

About RQ Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Chapter 2 Designing an RQ Experiment 7

Selecting the PCR Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Specifying the Components of an RQ Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Selecting the Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Selecting One- or Two-Step RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Choosing the Probes and Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Chapter 3 Performing Reverse Transcription 17

Guidelines for Preparing RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Converting Total RNA to cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Chapter 4 Generating Data from RQ Plates –

7300 or Standard 7500 System 21

Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Preparing the PCR Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Preparing the Reaction Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Creating a Relative Quantification (RQ) Plate Document . . . . . . . . . . . . . . . . . . . . . 25

Specifying Thermal Cycling Conditions and Starting the Run . . . . . . . . . . . . . . . . . 30

Analyzing and Viewing RQ Plate Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Exporting RQ Plate Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

vi Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide

Chapter 5 Generating Data from RQ Plates – 7500 Fast System 37

Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Preparing the PCR Master Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Preparing the Reaction Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Creating a Relative Quantification (RQ) Plate Document . . . . . . . . . . . . . . . . . . . . . 43

Specifying Thermal Cycling Conditions and Starting the Run . . . . . . . . . . . . . . . . . 48

Analyzing and Viewing RQ Plate Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Exporting RQ Plate Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Chapter 6 Analyzing Data in an RQ Study 59

Creating an RQ Study Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Configuring Analysis Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Adjusting the Baseline and Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Analyzing and Viewing the Results of the RQ Study . . . . . . . . . . . . . . . . . . . . . . . . 70

Reanalyzing an RQ Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Omitting Samples from a Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Exporting RQ Study Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Appendix A Creating Detectors 79

Appendix B Example RQ Experiment 81

References 89

Index 91

Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide vii

Preface

How to Use This Guide

Purpose of This

Guide

This guide is written for principal investigators and laboratory staff who conduct relative

quantification studies for gene expression using the Applied Biosystems

7300/7500/7500 Fast Real-Time PCR System.

Assumptions 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 drop-

down 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.

Preface

How to Use This Guide

viii Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide

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Refer to the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System

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7300/7500/7500 Fast Real-Time PCR System Site Preparation Guide for important

safety information.

Preface

How to Obtain More Information

Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide ix

How to Obtain More Information

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.

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