Data-driven normalization strategies for high-throughput quantitative RT-PCR.
BACKGROUND
High-throughput real-time quantitative reverse transcriptase polymerase chain reaction (qPCR) is a widely used technique in experiments where expression patterns of genes are to be profiled. Current stage technology allows the acquisition of profiles for a moderate number of genes (50 to a few thousand), and this number continues to grow. The use of appropriate normalization algorithms for qPCR-based data is therefore a highly important aspect of the data preprocessing pipeline.


RESULTS
We present and evaluate two data-driven normalization methods that directly correct for technical variation and represent robust alternatives to standard housekeeping gene-based approaches. We evaluated the performance of these methods against a single gene housekeeping gene method and our results suggest that quantile normalization performs best. These methods are implemented in freely-available software as an R package qpcrNorm distributed through the Bioconductor project.


CONCLUSIONS
The utility of the approaches that we describe can be demonstrated most clearly in situations where standard housekeeping genes are regulated by some experimental condition. For large qPCR-based data sets, our approaches represent robust, data-driven strategies for normalization.
Department of Biostatistics, Harvard School of Public Health, 677 Huntington Avenue, Boston, MA 02115, USA. jess@jimmy.harvard.edu
Jessica C
JC
Mar
Yasumasa
Y
Kimura
Kate
K
Schroder
Katharine M
KM
Irvine
Yoshihide
Y
Hayashizaki
Harukazu
H
Suzuki
David
D
Hume
John
J
Quackenbush
D000465:Algorithms; D019295:Computational Biology / Q000379:methods*; D030541:Databases, Genetic; D020869:Gene Expression Profiling; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*