Assays for determining poly(A) tail length and the polarity of mRNA decay in mammalian cells.
This chapter describes several methods for measuring the length of the mRNA poly(A) tail and a novel method for measuring mRNA decay. Three methods for measuring the length of a poly(A) tail are presented: the poly(A) length assay, the ligation-mediated poly(A) test (LM-PAT), and the RNase H assay. The first two methods are PCR-based assays involving cDNA synthesis from an oligo(dT) primer. The third method involves removing the poly(A) tail from the mRNA of interest. A major obstacle to studying the enzymatic step of mammalian mRNA decay has been the inability to capture mRNA decay intermediates with structural impediments such as the poly(G) tract used in yeast. To overcome this, we combined a standard kinetic analysis of mRNA decay with a tetracycline repressor-controlled reporter with an Invader RNA assay. The Invader RNA assay is a simple, elegant assay for the quantification of mRNA. It is based on signal amplification, not target amplification, so it is less prone to artifacts than other methods for nucleic acid quantification. It is also very sensitive, able to detect attomolar levels of target mRNA. Finally, it requires only a short sequence for target recognition and quantitation. Therefore, it can be applied to determining the decay polarity of a mRNA by measuring the decay rates of different portions of that mRNA.
Murray
Elizabeth L
EL
Department of Molecular and Cellular Biochemistry and the RNA Group, The Ohio State University, Columbus, Ohio, USA.
Schoenberg
Daniel R
DR
D000818:Animals; D006801:Humans; D008322:Mammals / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011061:Poly A / Q000032:analysis* / Q000235:genetics*; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D020871:RNA Stability
D011506:Proteins; D011061:Poly A