Polacek Denise C, Passerini Anthony G, Shi Congzhu, Francesco Nadeene M, Manduchi Elisabetta, Grant Gregory R, Powell Steven, Bischof Helen, Winkler Hans, Stoeckert Christian J, Davies Peter F
Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia 19104, USA.
Physiol Genomics. 2003 Apr 16;13(2):147-56. doi: 10.1152/physiolgenomics.00173.2002.
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.
尽管对于来自有限数量细胞和组织的微阵列分析而言，mRNA扩增是必要的，但扩增后转录谱的准确性尚未得到充分表征。我们在一个成熟的细胞因子[肿瘤坏死因子α（TNFα）]刺激的人内皮细胞体外模型中，使用13,824个人cDNA的滤膜阵列，测试了T7介导的转录线性扩增后差异基因表达的保真度。将扩增的反义RNA（aRNA）（来自100 ng总RNA，约1 ng mRNA）产生的转录谱与来自同一均匀样本池的未扩增RNA产生的转录谱进行比较。使用未扩增样本检测到的基因中，94%的基因通过扩增准确鉴定出TNFα诱导的差异表达。此外，使用扩增RNA鉴定出另外1150个基因可能存在差异表达，而使用未扩增RNA则未检测到这些基因。从该组中抽样的基因中，67%通过定量实时PCR验证为真正差异表达。因此，线性扩增除了证明相对于未扩增样本在基因表达上的保真度外，还提高了检测灵敏度，并增强了微阵列高通量筛选的发现潜力。
