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Real-time polymerase chain reaction-based exponential sample amplification for microarray gene expression profiling.

Analytical biochemistry (2005-01-15)
Zsolt B Nagy, János Z Kelemen, Liliána Z Fehér, Agnes Zvara, Kata Juhász, László G Puskás
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. Chi2 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.

MATERIALS
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Product Description

Sigma-Aldrich
Complete Whole Transcriptome Amplification Kit, DNA polymerase included, Complete Kit with optimized enzyme to amplify total RNA in <4 hours, no 3′ bias