ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3' RACE applications.

Amrei Jänicke, John Vancuylenberg, Peter R Boag, Ana Traven, Traude H Beilharz

RNA (New York, N.Y.) 2012 Jun

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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 Poly(A) Test (ePAT). The ePAT approach is as efficient as traditional Ligation-Mediated Poly(A) Test (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.

Citation

Amrei Jänicke, John Vancuylenberg, Peter R Boag, Ana Traven, Traude H Beilharz. ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3' RACE applications. RNA (New York, N.Y.). 2012 Jun;18(6):1289-95