title: Evaluation of bias associated with high-multiplex, target-specific pre-amplification.
abstract: We developed a novel PCR-based pre-amplification (PreAmp) technology that can increase the abundance of over 350 target genes one million-fold. To assess potential bias introduced by PreAmp we utilized ERCC RNA reference standards, a model system that quantifies measurement error in RNA analysis. We assessed three types of bias: amplification bias, dynamic range bias and fold-change bias. We show that our PreAmp workflow introduces only minimal amplification and fold-change bias under stringent conditions. We do detect dynamic range bias if a target gene is highly abundant and PreAmp occurred for 16 or more PCR cycles; however, this type of bias is easily correctable. To assess PreAmp bias in a gene expression profiling experiment, we analyzed a panel of genes that are regulated during differentiation using the NTera2 stem cell model system. We find that results generated using PreAmp are similar to results obtained using standard qPCR (without the pre-amplification step). Importantly, PreAmp maintains patterns of gene expression changes across samples; the same biological insights would be derived from a PreAmp experiment as with a standard gene expression profiling experiment. We conclude that our PreAmp technology can facilitate analysis of extremely limited samples in gene expression quantification experiments.
authors.affiliation: Reagents R&D, Life Science Group, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 9547, USA.
authors.forename: Steven T
authors.initials: ST
authors.lastname: Okino
authors.affiliation: Reagents R&D, Life Science Group, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 9547, USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Kong
authors.affiliation: Reagents R&D, Life Science Group, Bio-Rad Laboratories, 1329 Meyerside Drive, Mississauga, ON L5T 1C9, Canada.
authors.forename: Haya
authors.initials: H
authors.lastname: Sarras
authors.affiliation: Reagents R&D, Life Science Group, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 9547, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
keywords: Bias; ERCC; ERCC, external RNA controls consortium; Gene expression profiling; NIST, National Institute of Standards and Technology; NT2, NTera2; PCR; Pre-amplification; PreAmp; PreAmp, pre-amplification; RA, retinoic acid