ePAT: A simple method to tag adenylated RNA to measure poly(A)-tail length and other 3′ RACE applications
ePAT: A simple method to tag adenylated RNA to measure poly(A)-tail length and other 3′ RACE applications
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DOI：
10.1261/rna.031898.111
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发表时间：
2012-06-01
期刊：
影响因子：
4.5
通讯作者：
Beilharz, Traude H.
中科院分区：
文献类型：
--
作者：
Jaenicke, Amrei;Vancuylenberg, John;Beilharz, Traude H.
在RNA分子的3'末端添加poly（A）-尾影响稳定性、核输出和翻译效率。在细胞质中，poly（A）-尾长度的动态变化长期以来被认为是翻译沉默和激活之间的转换的反映。因此，在稳态下与任何给定mRNA相关的聚腺苷酸尾的测量可以用作其抑制状态的替代读数。在这里，我们描述了一种简单的新方法，利用大肠杆菌DNA聚合酶I的固有特性，使用DNA模板延伸RNA引物，在总RNA样品中标记3 '-腺苷酸化RNA。该标签可用作cDNA合成和随后的基因特异性PCR的锚以评估聚腺苷酸尾长度。我们称这种方法为扩展（P）under bar oly（（A）under bar）（T）under bar est（ePAT）。ePAT方法与传统的（L）在bar下连接-（M）在bar下介导（P）在bar下寡核苷酸（（A）在bar下）（T）在bar下测试（LM-PAT）测定一样有效，避免了与基于寡聚-dT的方法相关的内部引发问题，并且允许在3' RACE应用中准确分析聚（A）-尾长度和替代的3' UTR使用。
The addition of a poly(A)-tail to the 3' termini of RNA molecules influences stability, nuclear export, and efficiency of translation. In the cytoplasm, dynamic changes in the length of the poly(A)-tail have long been recognized as reflective of the switch between translational silence and activation. Thus, measurement of the poly(A)-tail associated with any given mRNA at steady-state can serve as a surrogate readout of its translation-state. Here, we describe a simple new method to 3'-tag adenylated RNA in total RNA samples using the intrinsic property of Escherichia coli DNA polymerase I to extend an RNA primer using a DNA template. This tag can serve as an anchor for cDNA synthesis and subsequent gene-specific PCR to assess poly(A)-tail length. We call this method extension (P) under bar oly((A) under bar) (T) under bar est (ePAT). The ePAT approach is as efficient as traditional (L) under bar igation-(M) under bar ediated (P) under bar oly((A) under bar) (T) under bar est (LM-PAT) assays, avoids problems of internal priming associated with oligo-dT-based methods, and allows for the accurate analysis of both the poly(A)-tail length and alternate 3' UTR usage in 3' RACE applications.