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J Biotechnol. 2007 May 10;129(4):628-34. Epub 2007 Feb 25.

Performance of different small sample RNA amplification techniques for hybridization on Affymetrix GeneChips.

Author information

1
RZPD German Resource Center for Genome Research, Heubnerweg 6, Berlin, Germany. f.wagner@rzpd.de

Abstract

A key issue in RNA amplification techniques is the preservation of original transcript abundance, however popular high-grade RNA amplification methods lack sufficient validation regarding the potential bias of gene expression profiles. This study evaluated a double-round T7-based and a PCR-based amplification protocol, using the Affymetrix GeneChip platform. Both small sample methods performed excellently in terms of yield and reproducibility (r>0.99), and also the within-method concordance with respect to differential gene expression was as high as with standard single-round T7-based amplification. However, when comparing the overlap of all differentially expressed genes between standard and small sample methods, this was only moderate for the double-round T7 (48.7-55.0%) as well as for the PCR-based amplification protocol (51.9-58.0%). In contrast, the concordance for the top 100 genes with highest fold changes was significantly higher, indicating that both small sample methods generate reliable results when focusing on strongly regulated genes.

PMID:
17408796
DOI:
10.1016/j.jbiotec.2007.02.015
[Indexed for MEDLINE]

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