Real-time polymerase chain reaction (PCR), also called quantitative PCR (qPCR), is one of the most powerful tools for nucleic acid analysis. However, inconsistencies in protocols, experimental design, sample processing and data interpretation can often affect results reproducibility and data reliability.
Establishing best practices is then essential to ensure accurate and meaningful outcomes. This handbook explores key strategies for optimizing qPCR workflows, from sample preparation to data analysis, helping you achieve more consistent and reliable results.
Access this handbook to explore:
- Essential factors to consider for experimental design and sample processing
- Best practices for nucleic acid extraction, reverse transcription and primer design
- Strategies for data validation and analysis to ensure reproducibility
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SKIP TO LESSON
Real-time PCR Handbook 3.0
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Handbook overview
Content summary
System compatibility
Module 1: Basics of real-time pcr
1.1 Introduction to real-time PCR
1.2 Overview of real-time PCR
1.3 Overview of real-time PCR components
1.4 Real-time PCR analysis technology
1.5 Real-time PCR fluorescence detection systems
1.6 Melting curve analysis
1.7 Passive reference dyes
1.8 Contamination prevention
1.9 Multiplex real-time PCR
1.10 Internal controls and reference genes
1.11 Real-time PCR instrument calibration
Module 1: Test your knowledge
MODULE 2: EXPERIMENTAL DESIGN
2.1 Introduction to experimental design
2.2 Real-time PCR assays
2.3 Amplicon and primer design
2.4 Nucleic acid purification and quantitation
2.5 Reverse transcription considerations
2.6 Experimental controls
2.7 Normalization methods
2.8 Standard Curves: reaction efficiency, sensitivity and reproducibility
2.9 Summary
Module 2: Test your knowledge
Module 3: Plate preparation
3.1 Reaction preparation and mixing
3.2 Plate loading
3.3 Plate sealing
3.4 Plate insertion
Module 3: Test your knowledge
Module 4: Data Analysis
4.1 Quantification methods
4.2 Quantitation with a standard curve
4.3 Comparative quantification
4.4 High-resolution melting curve analysis
4.5 Multiplex real-time PCR analysis
Module 5: Troubleshooting qpcr experiments
5.1 Troubleshooting - common considerations
5.2 Primer-dimers
5.3 Primer and probe storage
5.4 Real-time PCR inhibition and poor reaction efficiency
5.5 Software analysis settings
5.6 No amplification
5.7 Summary
FAQs
Module 6: Gene Expression Specificity
6.1 Introduction to gene expression analysis
6.2 Specificity challenges for gene expression analysis
6.3 Selecting your detection chemistry
6.4 TaqMan Assay specificity in gene expression analysis experiments
6.5 Using SYBR Green dye in gene expression analysis
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Content summary
This handbook was created to help you understand the principles of real-time PCR and best practices on all aspects of real-time PCR experiment design. The handbook consists of five modules plus additional resources to further assist you with your real-time PCR experiment.
We will continue to develop modules so we recommend that you bookmark this resource and check back for new content.
1
Basics of real-time PCR
2
Experimental design
3
Plate preparation
4
Data analysis
5
Troubleshooting
6
Gene Expression Specificity
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
© 2025 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan and AmpliTaq Gold are trademarks of Roche Molecular Systems, Inc., used under permission and license. Bioanalyzer is a trademark of Agilent Technologies Inc.Black Hole Quencher is a trademark of Biosearch Technologies, Inc. BLAST is a trademark of the National Library of Medicine. EvaGreen is a trademark of Biotium, Inc. iCycler, iCycler iQ, and MyiQ are trademarks of Bio-Rad Laboratories, Inc. LC Green is a trademark of BioFireDefense, LLC. LightCycler is a trademark of Roche Diagnostics GmbH. Rotor-Gene is a trademark of Qiagen GmbH.
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