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Guide to Performing Relative Quantitation of Gene Expression
Using Real-Time Quantitative PCR
Table of Contents
Section I: Introduction to Real-Time PCR and Relative Quantitation of Gene
Expression
1.
2.
3.
4.
Introduction
What is Relative Quantitation?
Terms and Acronyms
Relative Quantitation of Gene Expression Requires the Quantitation of Two
Different Genes (Target and Endogenous Control)
5. Factors Affecting Accurate Real-Time PCR Results
6. What is PCR Amplification Efficiency?
Section II: RNA Preparation and Reverse Transcription
1. Introduction
2. Quantifying Input RNA
3. Reverse Transcription (RT) for Relative Quantitation of Gene Expression
a. Two-step RT-PCR
b. One-step RT-PCR
4. Selecting Reverse Transcription and Real-Time PCR Reagents
5. Determination of Input RNA Amounts for a Relative Quantitation
Study
6. Identifying PCR Inhibition
7. How Much Genomic DNA Contamination can be Tolerated in a Relative
Quantitation of Gene Expression Assay?
Section III: Assay Selection and Design for Relative Quantitation
1.
2.
3.
4.
Selecting or Designing Primers and TaqMan® Probes for Relative Quantitation of
Gene Expression
TaqMan® Gene Expression Assays
Custom TaqMan® Gene Expression Assays
TaqMan® Pre-Developed Assay Reagents (TaqMan® PDARs)
Use of Primer Express® Software for the Design of Primer and Probe Sets for
Relative Quantitation of Gene Expression
Design of Assays for SYBR® Green I Applications
5.
Section IV. Identification and Selection of Endogenous Controls for Relative
Quantitation
1. Uniformity of Endogenous Control Expression.
2. Validation of Target and Control Genes for the Comparative CT Method
3. Multiplexing Endogenous Controls and Target Genes
Section V. Customized and Pre-Configured Relative Quantitation Gene Expression
A.
Products
1. TaqMan® Low Density Arrays (7900HT Microfluidic Cards)
2. Pre-Configured TaqMan® Low Density Arrays (Immune Profiling)
3. TaqMan® Cytokine Gene Expression Plate
4. TaqMan® Human Endogenous Control Plate
Section VI. Ordering Real Time PCR Reagents
2