pcrEfficiency: a Web tool for PCR amplification efficiency prediction.
BACKGROUND
Relative calculation of differential gene expression in quantitative PCR reactions requires comparison between amplification experiments that include reference genes and genes under study. Ignoring the differences between their efficiencies may lead to miscalculation of gene expression even with the same starting amount of template. Although there are several tools performing PCR primer design, there is no tool available that predicts PCR efficiency for a given amplicon and primer pair.


RESULTS
We have used a statistical approach based on 90 primer pair combinations amplifying templates from bacteria, yeast, plants and humans, ranging in size between 74 and 907 bp to identify the parameters that affect PCR efficiency. We developed a generalized additive model fitting the data and constructed an open source Web interface that allows the obtention of oligonucleotides optimized for PCR with predicted amplification efficiencies starting from a given sequence.


CONCLUSIONS
pcrEfficiency provides an easy-to-use web interface allowing the prediction of PCR efficiencies prior to web lab experiments thus easing quantitative real-time PCR set-up. A web-based service as well the source code are provided freely at http://srvgen.upct.es/efficiency.html under the GPL v2 license.
Genetics, Institute of Plant Biotechnology (IBV), Technical University of Cartagena (UPCT), Campus Muralla del Mar, 30202 Cartagena, Spain. izaskun.mallona@upct.es
Izaskun
I
Mallona
Julia
J
Weiss
Marcos
M
Egea-Cortines
D001419:Bacteria / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D020407:Internet; D015233:Models, Statistical; D010944:Plants / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D012984:Software
D017931:DNA Primers