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

Preview
Page 1
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System
Introduction and Example RQ Experiment
Relative Quantification Getting Started Guide
Designing an RQ 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 RQ Plates – 7300 or Standard 7500 System
FAST
Generating Data from RQ Plates – 7500 Fast System
Analyzing Data in an RQ 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 4378655 Rev. B 06/2010
Relative Quantification Experiment Workflow
Chapter 1
Introduction
About the 7300/7500/7500 Fast system
About Relative Quantification
About RQ Experiments
Chapter 2
Designing an RQ Experiment
Select the PCR method
Specify the components of an RQ 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 RQ Plate document
Program the thermal cycling conditions
Save the RQ Plate document
Start the run
View RQ plate data
Generating Data from RQ Plates Fast
Performing an RQ Study
3´ Reverse Primer
Oligo d(T) or random hexamer
Synthesis of 1st cDNA strand 3´
Generating Data from RQ Plates Standard
(OR)
Chapter 5
Select one-step or two-step RT-PCR
FAST
Create a new RQ Study document
Configure analysis settings
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Adjust the baseline and threshold
Analyze and view results
If necessary, omit samples
Export AQ Plate document, if desired
iii
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Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Contents
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
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
v
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
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Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Preface How to Use This Guide Purpose of This Guide Assumptions
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. 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 Relative 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 Relative 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 Absolute Quantification Getting Started Guide
4347825
4378656
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 Relative 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 Relative Quantification Getting Started Guide
Chapter 1
Introduction
About the 7300/7500/7500 Fast system
See page 2
About Relative Quantification
See page 2
About RQ Experiments
See page 3
Introduction
Designing an RQ Experiment
Primer Extended on mRNA 5´
3´ Reverse Primer
5´ cDNA
Oligo d(T) or random hexamer
Synthesis of 1st cDNA strand 3´
STANDARD TANDAR
Generating Data from RQ Plates Standard
5´ cDNA
Performing Reverse Transcription
OR
Performing an RQ Study
Generating Data from RQ Plates Fast
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative 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. The Applied Biosystems 7500 Fast Real-Time PCR System allows the user to perform high speed thermal cycling giving run times for quantitative real-time PCR applications (such as relative quantification) in fewer than 40 minutes.
Relative Quantification Assay
The 7300/7500/7500 Fast system allows the user to perform several assay types using plates or tubes in the 96-well format. This guide describes the relative quantification (RQ) assay type. For information about the other assay types, refer to the Real-Time PCR Systems Chemistry Guide (PN 4378658) and the Online Help for the 7300/7500/7500 Fast system (Online Help).
About Relative Quantification Real-time PCR Assays
RQ is performed using real-time PCR. In real-time PCR assays, 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. There are two types of quantitative real-time PCR: absolute and relative.
Definition
Relative quantification determines the change in expression of a nucleic acid sequence (target) in a test sample relative to the same sequence in a calibrator sample. The calibrator sample can be an untreated control or a sample at time zero in a time-course study (Livak and Schmittgen, 2001). For example, relative quantification is commonly used to compare expression levels of wild-type with mutated alleles or the expression levels of a gene in different tissues. RQ provides accurate comparison between the initial level of template in each sample, without requiring the exact copy number of the template. Further, the relative levels of templates in samples can be determined without the use of standard curves.
Notes
2
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Chapter 1 Introduction About RQ Experiments
About RQ Experiments 1 RQ Experiment Workflow
In this document, the term “RQ experiment” refers to the entire process of relative quantification, beginning with generating cDNA from RNA (reverse transcription) and ending with analyzing an RQ study. The RQ experiment workflow is shown on page iii.
RQ Studies with the 7300/7500/7500 Fast System
The data-collection part of an RQ assay is a single-plate document, called the RQ Plate. Amplification data from PCR runs is stored with sample setup information on the plate. The data-analysis part of an RQ assay is a multi-plate document, called the RQ Study. You can analyze up to ten RQ plates in a study. RQ Study documents neither control the instrument, nor do they provide tools for setting up or modifying plates. IMPORTANT! RQ Study software is an optional package for the 7300 instrument but it is
standard for the 7500 instrument and the 7500 Fast instrument. The following figure illustrates the RQ Study process.
Liver
liverplate.sds
Kidney kidneyplate.sds
Bladder
bladderplate.sds
Reaction Plate
7300/7500/ 7500 Fast System
SDS Software
RQ Plate Documents
SDS Software
RQ Study Document
Note: The 7300/7500/7500 Fast system software uses the comparative method (∆∆CT)
to calculate relative quantities of a nucleic acid sequence. If you want to perform relative quantification using the relative standard curve method, you should use an AQ assay type and consult the Real-Time PCR Systems Chemistry Guide for details on how to set up a run and analyze this type of assay.
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
3
Chapter 1 Introduction About RQ 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 ∆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.
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 signal that is an indicator of 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).
The figure below shows a representative amplification plot and includes some of the terms defined in the previous table. 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 Relative Quantification Getting Started Guide
Chapter 1 Introduction About RQ Experiments
Required User-Supplied 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)
MicroAmp® Optical 96-Well Reaction Plate
Applied Biosystems - (PN 4306757)
Optical 96-Well Fast Thermal Cycling Plate with Barcode (code 128)
Applied Biosystems - (PN 4346906)
Optical Adhesive Cover
Applied Biosystems- (PN 4311971)
1
Labeled primers and probes from one of the following sources: • TaqMan® Gene Expression Assays (predesigned primers and probes) • TaqMan® Custom Gene Expression Assays service (predesigned primers and probes)
• Applied Biosystems Web site
• 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
Safety goggles
MLS
Vortexer
MLS
Example RQ Experiment To better illustrate how to design, perform, and analyze RQ experiments, this section guides you through an example experiment. The example experiment represents a typical RQ experiment setup that you can use as a quick-start procedure to familiarize yourself with the RQ workflow. Detailed steps in the RQ workflow are described in the
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
5
Chapter 1 Introduction About RQ Experiments
subsequent chapters of this guide. Also in the subsequent chapters are Example Experiment boxes that provide details for some of the related steps in the example experiment. Refer to Appendix B, “Example RQ Experiment,” on page 81 for more information.
Notes
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Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Chapter 2
Designing an RQ Experiment
Introduction and Example RQ Experiment
Designing an RQ Experiment
Primer Extended on mRNA 5´
3´ Reverse Primer
5´ cDNA
Oligo d(T) or random hexamer
Synthesis of 1st cDNA strand 3´
STANDARD TANDAR
Generating Data from RQ Plates Standard
5´ cDNA
Performing Reverse Transcription
OR
Performing an RQ Study
Generating Data from RQ Plates Fast
Select the PCR method
See page 8
Specify the components of an RQ experiment
See page 9
Select the chemistry
See page 11
Select one-step or two-step RT-PCR
See page 12
Choose the probes and primers
See page 14
2
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
7
Chapter 2 Designing an RQ Experiment Selecting the PCR Method
Selecting the PCR Method Types of PCR Methods
PCR is performed as either of the following: • A singleplex reaction, where a single primer pair is present in the reaction tube or well. Only one target sequence or endogenous control can be amplified per reaction. • A multiplex reaction, where two or more primer pairs are present in the reaction. Each primer pair amplifies either a target sequence or an endogenous control. Target Primer Set Endogenous Control Primer Set Singleplex PCR
Selection Criteria
Multiplex PCR
cDNA GR2331
Both methods give equivalent results for relative quantification experiments. To select a method, consider the: • Type of chemistry you use to detect PCR products – Singleplex PCR can use either SYBR® Green or TaqMan® reagent-based chemistry. Multiplex PCR can use only TaqMan reagent-based chemistry. • Amount of time you want to spend optimizing and validating your experiment – Amplifying target sequences and endogenous controls in separate reactions (singleplex PCR) requires less optimization and validation than multiplex PCR. Among the factors to consider in multiplex PCR are primer limitation, the relative abundance of the target and reference sequences (the endogenous control must be more abundant than the targets), and the number of targets in the study. IMPORTANT! As the number of gene targets increases, the singleplex format is typically
more effective than the multiplex format because less optimization is required.
Additionally, running multiple reactions in the same tube increases throughput and reduces the effects of pipetting errors. For more information about multiplex and singleplex PCR, refer to the Real-Time PCR Systems Chemistry Guide (PN 4343978). Sample Experiment The singleplex PCR method is used in the example experiment because: • The number of targets to be amplified (23 genes, plus one endogenous control) is large • Optimization and validation requirements are reduced for singleplex experiments
Notes
8
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Chapter 2 Designing an RQ Experiment Specifying the Components of an RQ Experiment
Specifying the Components of an RQ Experiment After you decide to use the singleplex or multiplex method, specify the required components of the RQ experiment for every sample: • A target – The nucleic acid sequence that you are studying. • A calibrator – The sample used as the basis for comparative results. • An endogenous control – A gene present at a consistent expression level in all experimental samples. By using an endogenous control as an active reference, you can normalize quantification of a cDNA target for differences in the amount of cDNA added to each reaction. Note that: – Each sample type (for example, each tissue in a study comparing multiple tissues) requires an endogenous control. – If samples are spread across multiple plates, each plate must have an endogenous control. Additionally, every plate must include an endogenous control for every sample type on the plate. Typically, housekeeping genes such as β-actin, glyceraldehyde-3-phosphate (GAPDH), and ribosomal RNA (rRNA) are used as endogenous controls, because their expression levels tend to be relatively stable. • Replicate wells – For relative quantification studies, Applied Biosystems recommends the use of three or more replicate reactions per sample and endogenous control to ensure statistical significance. For more information about these requirements, refer to the Real-Time PCR Systems Chemistry Guide.
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
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2
Chapter 2 Designing an RQ Experiment Specifying the Components of an RQ Experiment
Sample Experiment In the example experiment, the objective is to compare the expression levels of several genes in the liver, kidney, and bladder tissue of an individual. The 23 genes of interest, including ACVR1, ACVR2, CCR2, CD3D, and FLT4, are the targets and the liver samples serve as the calibrator. The SDS Software 1.3.1 sets gene expression levels for the calibrator samples to 1. Consequently, if more ACRV1 is in the kidney than in the liver, the gene expression level of ACRV1 in the kidney is greater than 1. Similarly, if less CD3D is in the bladder than in the liver, the gene expression level of CD3D in the bladder is less than 1. Because RQ is based on PCR, the more template in a reaction, the more the PCR product and the greater the fluorescence. To adjust for possible differences in the amount of template added to the reaction, GAPDH serves as an endogenous control. (Expression levels of the endogenous control are subtracted from expression levels of target genes.) An endogenous control is prepared for each tissue. The experiment includes three sets of endogenous controls-one for each tissue. Also, the endogenous control for each tissue must be amplified on the same plate as the target sequences for that tissue. Finally, note that the experiment uses the singleplex PCR format, and therefore, the endogenous controls are amplified in wells different from the target wells. Four replicates of each sample and endogenous control are performed to ensure statistical significance (see below).
Note: The example RQ experiment requires a separate plate for each of the three tissues because of the large number of genes being studied. You can also design experiments so that several samples are amplified on the same plate, as shown in the following table. Singleplex Assay
Multiplex Assay
In the example RQ experiment, each plate contains a single sample type (tissue). The endogenous control for each tissue is on the same plate as the targets for that tissue.
If the example experiment were run with multiple sample types on the same plate, an endogenous control for each sample type must also be included on the same plate, as shown here. Liver
GR2323 Regulus Liver_96Plate.eps
Liver samples
Kidney
Bladder
GR2322 Regulus LivKidBladPlate.eps
Samples
Endogenous controls (GAPDH) GR2323
GR2324 Regulus Kidney_96Plate.eps
GR2322
Endogenous controls (GAPDH)
Kidney samples
Endogenous controls (GAPDH) GR2324
GR2325 Regulus Bladder_96Plate.eps
Bladder samples
Endogenous controls (GAPDH) GR2325
Notes
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Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide