Performance of different small sample RNA amplification techniques for hybridization on Affymetrix GeneChips.
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.
Wagner
Florian
F
RZPD German Resource Center for Genome Research, Heubnerweg 6, Berlin, Germany. f.wagner@rzpd.de
Radelof
Uwe
U
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
D012334:RNA, Neoplasm