Specifying The Components Of An Rq Experiment - Applied Biosystems 7500 Getting Started Manual

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:
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
• 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.
Chapter 2 Designing an RQ Experiment
Specifying the Components of an RQ Experiment
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