Assaying the polyadenylation state of mRNAs
Assaying the polyadenylation state of mRNAs
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DOI：
10.1006/meth.1998.0705
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发表时间：
1999-01-01
期刊：
影响因子：
--
通讯作者：
Strickland, S
中科院分区：
文献类型：
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作者：
Sallés, FJ;Richards, WG;Strickland, S
在大多数真核生物mRNA的3'末端存在的poly（A）尾可以在信息翻译和稳定性中起关键作用。因此，鉴定poly（A）尾长度的改变可以对mRNA的功能和随后的生理影响产生重要的见解。在这里，我们提出了三种方法来测定一个特定的mRNA的总细胞RNA的背景下的聚腺苷酸化。所述的第一种方法，寡聚（dT）/RNA酶H-Northern分析，是用于聚腺苷酸化的经典劳动密集型测定，并被纳入作为历史参考，并作为随后所述的聚（A）试验（PAT）测定的潜在实验对照。PAT方法-cDNA末端快速扩增-PAT（RACE-PAT）和连接酶介导的PAT（LM-PAT）-是聚合酶链反应驱动的检测方法，可实现速度、灵敏度和长度定量。PAT测定可以在一天内进行，并且可以很容易地检测亚纳克量的总细胞RNA中存在的mRNA的poly（A）状态。(C)北京：科学出版社.
The poly(A) tail present at the 3' end of most eukaryotic mRNAs can play a critical role in message translation and stability. Therefore, identifying alterations in poly(A) tail length can yield important insights into an mRNA's function and subsequent physiological impact. Here, we present three methods for assaying polyadenylation of a specific mRNA in the context of total cellular RNA. The first method described, oligo(dT)/RNase H-Northern analysis, is the classic labor-intensive assay for polyadenylation and is included for historical reference and as a potential experimental control for the poly(A) test (PAT) assays described subsequently. The PAT methods-rapid amplification of cDNA ends-PAT (RACE-PAT), and ligase-mediated PAT (LM-PAT)-are polymerase chain reaction-driven assays that allow speed, sensitivity, and length quantitation. The PAT assays can be conducted in a single day and can readily detect the poly(A) status of an mRNA present in subnanogram quantities of total cellular RNA. (C) 1999 Academic Press.