Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA

Abstract

Although mRNA amplification is necessary for microarray analyses from limited amounts of cells and tissues, the accuracy of transcription profiles following amplification has not been well characterized. We tested the fidelity of differential gene expression following linear amplification by T7-mediated transcription in a well-established in vitro model of cytokine [tumor necrosis factor alpha (TNFalpha)]-stimulated human endothelial cells using filter arrays of 13,824 human cDNAs. Transcriptional profiles generated from amplified antisense RNA (aRNA) (from 100 ng total RNA, approximately 1 ng mRNA) were compared with profiles generated from unamplified RNA originating from the same homogeneous pool. Amplification accurately identified TNFalpha-induced differential expression in 94% of the genes detected using unamplified samples. Furthermore, an additional 1,150 genes were identified as putatively differentially expressed using amplified RNA which remained undetected using unamplified RNA. Of genes sampled from this set, 67% were validated by quantitative real-time PCR as truly differentially expressed. Thus, in addition to demonstrating fidelity in gene expression relative to unamplified samples, linear amplification results in improved sensitivity of detection and enhances the discovery potential of high-throughput screening by microarrays.

Coexisting Pro-Inflammatory and Anti-Oxidative Endothelial Transcription Profiles in a Disturbed Flow Region of the Adult Porcine Aorta

“…The validation of about two of three genes after linear amplification is consistent with our previous report, for which we used a model system (26).…”

“…We have previously evaluated the linear amplification protocol by using similar amounts of human matched probe͞target and found it to identify differential expression with fidelity and enhanced sensitivity (26). In agreement with this previous study, the QRT-PCR results reported in the current study corroborate approximately two of three genes tested.…”

“…In particular, the two may share common mechanisms mediated by NF-B. We have previously profiled EC gene expression in response to TNF-␣ in vitro (26), and we find the overlap with the present in vivo study to be small. Up-regulated genes common to both studies included monocyte chemotactic protein 1, interleukin 1␣, NF-B1, NF-B2, NF-B inhibitor ␣, TNF-␣-induced protein 6, and cycloxygenase-2, whereas common down-regulated genes included TNF receptorassociated factor 6 and the chemokine receptor CXCR4.…”

“…Image files were quantified with ARRAYVISION 6.3 (Imaging Research, St. Catherine's, ON, Canada) as described (26). A putative set of differentially expressed genes was identified by taking the union of predictions made through the methods of ARRAYSTAT Quantitative real-time PCR (QRT-PCR) was performed on selected genes as described (26). Expression was measured in paired DF and UF samples from six animals, and a ratio was calculated for each animal based on a minimum of three replicate observations.…”

Genome-Wide Expression Studies of Atherosclerosis

“…Pooling of samples limits the possibilities for downstream statistical analysis (see "Key Features of Gene Expression Studies in Atherosclerosis"). Pros and cons of amplification have been discussed in several recent articles and include issues such as reproducibility 29 and effects on magnitude of differential expression 30 caused by amplification. An alternative approach to obtain relatively pure cell populations and to remove cell products is to culture cells after isolation from entire vessel wall samples.…”