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doi:10.1016/j.ab.2004.09.044 
Copyright © 2004 Elsevier Inc. All rights reserved.
Real-time polymerase chain reaction-based exponential sample amplification for microarray gene expression profiling
Zsolt B. Nagya, János Z. Kelemena, Liliána Z. Fehéra, Ágnes Zvaraa, Kata Juhászb and László G. Puskása, 
, 
Received 2 August 2004.
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Abstract
Conventional approaches to target labeling for gene expression analysis using microarray technology typically require relatively large amounts of RNA, a serious limitation when the available sample is limited. Here we describe an alternative exponential sample amplification method by using quantitative real-time polymerase chain reaction (QRT-PCR) to follow the amplification and eliminate the overamplified cDNA which could distort the quantitative ratio of the starting mRNA population. Probes generated from nonamplified, PCR-amplified, and real-time-PCR-amplified cDNA samples were generated from lipopolysaccharide-treated and nontreated mouse macrophages and hybridized to mouse cDNA microarrays. Signals obtained from the three protocols were compared. Reproducibility and reliability of the methods were determined. The Pearson correlation coefficients for replica experiments were r = 0.927 and r = 0.687 for QRT-PCR-amplification and PCR-overamplification protocols, respectively. χ2 test showed that overamplification resulted in major biases in expression ratios, while these alterations could be eliminated by following the cycling status with QRT-PCR. Our exponential sample amplification protocol preserves the original expression ratios and allows unbiased gene expression analysis from minute amounts of starting material.
Keywords: Exponential sample amplification; Quantitative real-time PCR; Microarray; Gene expression profiling
Article Outline
- Materials and methods
- Cell culture, RNA isolation, and quality assessment
- Preparation of cDNA targets for signal amplification
- Preparation of PCR-amplified and PCR-overamplified labeled probes
- Array hybridization and data analysis
- Quantitative real-time PCR
- Results and discussion
- Acknowledgements
- References
