title: Performance of different small sample RNA amplification techniques for hybridization on Affymetrix GeneChips.
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.
authors.affiliation: RZPD German Resource Center for Genome Research, Heubnerweg 6, Berlin, Germany. f.wagner@rzpd.de
authors.forename: Florian
authors.initials: F
authors.lastname: Wagner
authors.forename: Uwe
authors.initials: U
authors.lastname: Radelof
mesh_terms: D001709:Biotechnology; D005784:Gene Amplification; D006367:HeLa Cells; D006801:Humans; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012334:RNA, Neoplasm / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: D012334:RNA, Neoplasm