High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay
High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay
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DOI：
10.1261/rna.1136108
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发表时间：
2008-09-01
期刊：
影响因子：
4.5
通讯作者：
Koszinowski, Ulrich H.
中科院分区：
文献类型：
--
作者：
Doelken, Lars;Ruzsics, Zsolt;Koszinowski, Ulrich H.
细胞中的RNA水平由RNA合成和衰变的相对速率决定。最先进的转录分析仅采用总细胞RNA。因此，RNA水平的变化不能归因于RNA合成或衰变，并且时间分辨率较差。最近，据报道，新转录的RNA可以使用巯基化核苷生物合成标记1-2小时，从总细胞RNA中纯化并进行微阵列分析。然而，为了在分子水平上研究信号事件，需要分析几分钟内发生的变化。我们开发了一种改进的方法，在10-15分钟的代谢标记后将总细胞RNA分离成新转录的和预先存在的RNA。采用新的计算工具进行阵列归一化和半衰期测定，我们同时研究短期RNA合成和衰变以及它们对细胞转录水平的影响。作为一个例子，我们研究了成纤维细胞对I型和II型干扰素（IFN）的反应。在干扰素受体激活的前三个小时内，在15-30分钟内不同时间转录的RNA的分析导致微阵列灵敏度增加>10倍，并提供了IFN介导的基因表达变化的动力学的综合概况。我们确定了一个以前未公开的高度连接的网络的短暂的成绩单选择性下调IFN γ在30和60分钟之间IFN治疗后表现出强烈的关联与细胞周期和凋亡，表明新的机制，IFN γ影响这些途径。
RNA levels in a cell are determined by the relative rates of RNA synthesis and decay. State-of-the-art transcriptional analyses only employ total cellular RNA. Therefore, changes in RNA levels cannot be attributed to RNA synthesis or decay, and temporal resolution is poor. Recently, it was reported that newly transcribed RNA can be biosynthetically labeled for 1-2 h using thiolated nucleosides, purified from total cellular RNA and subjected to microarray analysis. However, in order to study signaling events at molecular level, analysis of changes occurring within minutes is required. We developed an improved approach to separate total cellular RNA into newly transcribed and preexisting RNA following 10-15 min of metabolic labeling. Employing new computational tools for array normalization and half-life determination we simultaneously study short-term RNA synthesis and decay as well as their impact on cellular transcript levels. As an example we studied the response of fibroblasts to type I and II interferons (IFN). Analysis of RNA transcribed within 15-30 min at different times during the first three hours of interferon-receptor activation resulted in a >10-fold increase in microarray sensitivity and provided a comprehensive profile of the kinetics of IFN-mediated changes in gene expression. We identify a previously undisclosed highly connected network of short-lived transcripts selectively down-regulated by IFN gamma in between 30 and 60 min after IFN treatment showing strong associations with cell cycle and apoptosis, indicating novel mechanisms by which IFN gamma affects these pathways.