Fidelity and enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA.
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.
Polacek
Denise C
DC
Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia 19104, USA.
Passerini
Anthony G
AG
Shi
Congzhu
C
Francesco
Nadeene M
NM
Manduchi
Elisabetta
E
Grant
Gregory R
GR
Powell
Steven
S
Bischof
Helen
H
Winkler
Hans
H
Stoeckert
Christian J
CJ
Davies
Peter F
PF
D015982:Bias; D002460:Cell Line; D030541:Databases, Genetic; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D020407:Internet; D036103:Nanotechnology / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism*; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha