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

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 read-out of its translation-state. Here we describe a simple new method to 3a??-tag adenylated RNA in total RNA samples using the intrinsic property of E. 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.