期刊
RNA
卷 18, 期 6, 页码 1289-1295出版社
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.031898.111
关键词
mRNA polyadenylation; alternative polyadenylation; translational control; LM-PAT assay
资金
- Australian Research Council [DP0878224]
- NHMRC [606575]
- ARC [DP1092850]
- Australian Research Council [DP1092850, DP0878224] Funding Source: Australian Research Council
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.
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