Appendix B
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide 81
Notes
B
Example RQ Experiment
Overview To better illustrate how to design, perform, and analyze RQ experiments, this section
guides you through an example experiment. The example experiment represents a
typical RQ experiment setup that you can use as a quick-start procedure to familiarize
yourself with the RQ workflow. Detailed steps in the RQ workflow are described in the
subsequent chapters of this guide. Also in the subsequent chapters are Example
Experiment boxes that provide details for some of the related steps in the example
experiment.
Description The objective of the example RQ experiment is to compare the levels of expression of 23
genes in the liver, kidney, and bladder tissue of an individual.
The experiment is designed for singleplex PCR: Samples and endogenous controls were
amplified in separate wells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
serves as the endogenous control. Four replicates of each sample and endogenous control
are amplified. (In this experiment, an entire 96-well-plate is devoted to each tissue
because the four replicates of each of the 23 genes plus the endogenous control require
all 96 wells.)
Primer/probe sets are selected from the Applied Biosystems TaqMan
®
Gene Expression
Assays product line.
Reactions are set up for two-step RT-PCR, where the High Capacity cDNA Archive Kit
and the TaqMan
®
Universal PCR Master Mix are used for reverse transcription and
PCR, respectively.
Data are generated by running three RQ plates, one for each tissue.
All three plates are analyzed in an RQ study, with the liver samples serving as the
calibrator.
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