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 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.
RNA