title: ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3' RACE applications.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Amrei
authors.initials: A
authors.lastname: Jänicke
authors.forename: John
authors.initials: J
authors.lastname: Vancuylenberg
authors.forename: Peter R
authors.initials: PR
authors.lastname: Boag
authors.forename: Ana
authors.initials: A
authors.lastname: Traven
authors.forename: Traude H
authors.initials: TH
authors.lastname: Beilharz
mesh_terms: D000818:Animals; D004256:DNA Polymerase I / Q000737:chemistry; D005821:Genetic Techniques; D009838:Oligodeoxyribonucleotides / Q000032:analysis; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis*
chemical_list: D009838:Oligodeoxyribonucleotides; C034477:RNA primers; D012333:RNA, Messenger; C027903:oligo (dT); D012313:RNA; D004256:DNA Polymerase I