Kenzelmann M, Maertens S, Hergenhahn M, Kueffer S, Hotz-Wagenblatt A, Li L, Wang S, Ittrich C, Lemberger T, Arribas R, Jonnakuty S, Hollstein M C, Schmid W, Gretz N, Gröne H J, Schütz G
Department of Molecular Biology of the Cell I, German Cancer Research Center, 69120 Heidelberg, Germany.
Proc Natl Acad Sci U S A. 2007 Apr 10;104(15):6164-9. doi: 10.1073/pnas.0610439104. Epub 2007 Apr 3.
Current methods to analyze gene expression measure steady-state levels of mRNA. To specifically analyze mRNA transcription, we have developed a technique that can be applied in vivo in intact cells and animals. Our method makes use of the cellular pyrimidine salvage pathway and is based on affinity-chromatographic isolation of thiolated mRNA. When combined with data on mRNA steady-state levels, this method is able to assess the relative contributions of mRNA synthesis and degradation/stabilization. It overcomes limitations associated with currently available methods such as mechanistic intervention that disrupts cellular physiology, or the inability to apply the techniques in vivo. Our method was first tested in serum response of cultured fibroblast cells and then applied to the study of renal ischemia reperfusion injury, demonstrating its applicability for whole organs in vivo. Combined with data on mRNA steady-state levels, this method provided a detailed analysis of regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level.
当前分析基因表达的方法是测量mRNA的稳态水平。为了特异性地分析mRNA转录，我们开发了一种可在完整细胞和动物体内应用的技术。我们的方法利用细胞嘧啶补救途径，基于硫醇化mRNA的亲和色谱分离。当与mRNA稳态水平的数据相结合时，该方法能够评估mRNA合成与降解/稳定化的相对贡献。它克服了与现有方法相关的局限性，如破坏细胞生理的机械干预，或无法在体内应用这些技术。我们的方法首先在培养的成纤维细胞的血清反应中进行测试，然后应用于肾缺血再灌注损伤的研究，证明了其在体内对整个器官的适用性。结合mRNA稳态水平的数据，该方法提供了对mRNA表达调控机制以及不同途径中RNA合成和周转的相对贡献的详细分析，并鉴定了转录水平低丰度表达的基因。
