( CATGTTC- 3' Operon) ...)Real-Time Multiplex PCR from Genomic DNA Using the iCycler iQ Detection System

CATGTTC- 3', Operon)

0.15 l 100 M GAPDH reverse primer (5'-CTCGCTCCTGGAAGATG-3', Operon)

0.1 l 100 M GAPDH probe (5'-FAM-CGGCACAGTCAAGGCCGAGAATGGTGCCG- DABCYL-3', Operon)

1.0 l plasmid DNA dilution

20.2 l PCR-grade ddH₂0

PCR conditions were 3 min at 95C, followed by 50 cycles of 10 sec at 95C, 45 sec at 55C.

Interpretation of Results
Gene expression studies of multiple gene targets require both precise and accurate quantitation of the same samples at the same time. The iCycler iQ system displays powerful multiplexing capabilities that we demonstrate here on genomic and plasmid targets. Furthermore, we demonstrate the need to optimize PCR efficiency when working with varying starting template concentrations.

In order to conduct successful multiplexing you must (1) maximize and equalize the efficiency of each reaction and (2) minimize any cross-reactivity between individual reactions. Maximizing the efficiency of a reaction allows accurate quantitation over a wider range of starting template concentrations and improves the reproducibility of replicate samples. It is equally important that the individual reactions have similar efficiencies because any differences in individual efficiencies will be amplified when the components of the 2 reactions are combined. If you know that the multiplex templates differ in concentration by less than 1,000-fold, then you can accept efficiency differences of about 10%. For example, one reaction may be 96% and the other 87% efficient. If you do not know the template concentration difference, or if it is gre
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