Using Klenow-Mediated Extension to Measure Poly(A)-Tail Length and Position in the Transcriptome

  • Man Chun Lee
  • Amrei Jänicke
  • Traude Helene Beilharz
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1125)

Abstract

The poly(A)-tail that terminates most mRNA and many noncoding RNA is a convenient “hook” to isolate mRNA. However the length of this tail and its position within the primary RNA transcript can also hold diagnostic value for RNA metabolism. In general, mRNA with a long poly(A)-tail is well translated, whereas a short poly(A)-tail can indicate translational silencing. A short poly(A)-tail is also appended to RNA-decay intermediates via the TRAMP complex. A number of approaches have been developed to measure the length and position of the poly(A)-tail. Here, we describe a simple method to “tag” adenylated RNA using the native function of DNA polymerase I to extend an RNA primer on a DNA template in second-strand DNA synthesis. This function can be harnessed as a means to purify, visualize, and quantitate poly(A)-dynamics of individual RNA and the transcriptome en masse.

Key words

ePAT End labeling Polyadenylation Poly(A)-tail length Klenow polymerase Translational control 

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Man Chun Lee
    • 1
  • Amrei Jänicke
    • 1
  • Traude Helene Beilharz
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyMonash University AustraliaMelbourneAustralia

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