Selecting The Pcr Method; Chapter 2 Designing An Rq Experiment - Applied Biosystems 7500 Getting Started Manual

Chapter 2 Designing an RQ Experiment

Selecting the PCR Method

Selecting the PCR Method
Types of PCR
Methods
Selection Criteria
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
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.
Singleplex PCR
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
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.
As the number of gene targets increases, the singleplex format is typically
IMPORTANT!
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).
Multiplex PCR
reagent-based chemistry. Multiplex PCR can use only
®
Target Primer Set
Endogenous Control
Primer Set
cDNA
GR2331