Using Klenow-mediated extension to measure poly(A)-tail length and position in the transcriptome

Man Chun Lee, Amrei Jaenicke, Traude Helene Beilharz

Research output: Contribution to journalArticleResearchpeer-review

2 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)25 - 42
Number of pages18
JournalMethods in Molecular Biology
Volume1125
DOIs
Publication statusPublished - 2014

Cite this

@article{2f8b84a675bf40d6b4e75ea29d377122,
title = "Using Klenow-mediated extension to measure poly(A)-tail length and position in the transcriptome",
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.",
author = "Lee, {Man Chun} and Amrei Jaenicke and Beilharz, {Traude Helene}",
year = "2014",
doi = "10.1007/978-1-62703-971-0_3",
language = "English",
volume = "1125",
pages = "25 -- 42",
journal = "Methods in Molecular Biology",
issn = "1064-3745",
publisher = "Humana Press",

}

Using Klenow-mediated extension to measure poly(A)-tail length and position in the transcriptome. / Lee, Man Chun; Jaenicke, Amrei; Beilharz, Traude Helene.

In: Methods in Molecular Biology, Vol. 1125, 2014, p. 25 - 42.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Using Klenow-mediated extension to measure poly(A)-tail length and position in the transcriptome

AU - Lee, Man Chun

AU - Jaenicke, Amrei

AU - Beilharz, Traude Helene

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

UR - http://download.springer.com/static/pdf/457/chp%253A10.1007%252F978-1-62703-971-0_3.pdf?auth66=1425594776_a2b565c658a252c6218a10df560c0ee1&ext=.pdf

U2 - 10.1007/978-1-62703-971-0_3

DO - 10.1007/978-1-62703-971-0_3

M3 - Article

VL - 1125

SP - 25

EP - 42

JO - Methods in Molecular Biology

JF - Methods in Molecular Biology

SN - 1064-3745

ER -