Andus T, Targan S R, Deem R, Toyoda H
Department of Internal Medicine I, University of Regensburg, Germany.
Reg Immunol. 1993 Jan-Feb;5(1):11-7.
We have developed a method to quantitate TNF alpha-mRNA in small numbers of cells by reverse transcription followed by competitive polymerase chain reaction (RT-PCR). RT-PCR allowed the accurate quantitation of TNF alpha-mRNA over a 1000-fold range of concentration. The recovery of RNA isolated from 1000 to 10,000 cells was optimized by reducing sample volumes and by adding 1 microgram yeast RNA. Using these modifications we accurately measured TNF alpha-mRNA in as little as 10,000 U937 cells by RT-PCR. Then we measured TNF alpha-mRNA in lamina propria mononuclear cells isolated from uninflamed and inflamed colonic mucosa from patients with inflammatory bowel disease (IBD). A 9-fold increase (5.4 copies per cell) was found in mononuclear cells from the gut of the inflamed regions compared to those cells from the uninflamed regions (0.6 copies per cell). These findings demonstrated the utility of this method in measuring differences of expression of the TNF-alpha gene in small number of cells isolated from tissues.
我们开发了一种方法，通过逆转录随后进行竞争性聚合酶链反应（RT-PCR）来定量少量细胞中的肿瘤坏死因子α（TNFα）-mRNA。RT-PCR能够在1000倍的浓度范围内准确地定量TNFα-mRNA。通过减少样品体积并添加1微克酵母RNA，优化了从1000至10000个细胞中分离RNA的回收率。使用这些改进方法，我们通过RT-PCR在低至10000个U937细胞中准确测量了TNFα-mRNA。然后，我们测量了从炎症性肠病（IBD）患者的未发炎和发炎结肠黏膜中分离出的固有层单核细胞中的TNFα-mRNA。与未发炎区域的细胞（每个细胞0.6个拷贝）相比，发炎区域肠道中的单核细胞中发现增加了9倍（每个细胞5.4个拷贝）。这些发现证明了该方法在测量从组织中分离出的少量细胞中TNF-α基因表达差异方面的实用性。
