Chapter 2 Designing an RQ Experiment
Choosing the Probes and Primers
14 Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
Notes
Choosing the Probes and Primers
Choose probe and primer sets for both your target and endogenous control sequences.
Applied Biosystems provides three options for choosing primers and probes:
• TaqMa n
®
Gene Expression Assays – Provide you with optimized, ready-to-use
TaqMan reagent-based 5′ -nuclease assays for human, mouse, or rat transcripts. For
information on available primer/probe sets, go to:
http://www.allgenes.com
• TaqMa n
®
Custom Gene Expression Assays – Designs, synthesizes, formulates,
and delivers quality-controlled primer and probe sets. Use this service if the assay
you need is not currently available. To place an order, contact your Applied
Biosystems representative or go to http://www.allgenes.com.
• Primer Express
®
Software – Helps you design primers and probes for your own
quantification assays. For more information about using this software, refer to the
Primer Express Software v3.0 Getting Started Guide (PN 4362460).
Applied Biosystems provides assay design guidelines that have been developed
specifically for quantification assays. When followed, these guidelines provide a
reliable system for assay design and optimization. For information about the assay
design guidelines, refer to the Real-Time PCR Systems Chemistry Guide.
If you ordered TaqMan
®
Gene Expression Assays or TaqMan
®
Custom Gene Expression
Assays, probes are already labeled with FAM
™
as a reporter dye. If you design your own
assays, you need to specify a reporter dye for your custom probe(s). For singleplex
experiments, you can use the same dye for targets and endogenous control(s). For
multiplex experiments, the probe for the target is typically labeled with FAM dye and
that for the endogenous control with VIC
®
dye.
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