Preparation of Poly(A)+ RNA

Robert E. Kingston1

1 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.5
DOI:  10.1002/0471142727.mb0405s21
Online Posting Date:  May, 2001
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Abstract

Most messenger RNAs contain a poly(A) tail, while structural RNAs do not. Poly(A) selection therefore enriches for messenger RNA. The technique has proved essential for construction of cDNA libraries. It is also useful when analyzing the structure of low‐abundance mRNAs. Removing the ribosomal and tRNAs from a preparation increases the amount of RNA that can be clearly analyzed by S1 analysis, for example, thus allowing detection of a low level message. This protocol separates poly(A)+ RNA from the remainder of total RNA, which is largely rRNA and tRNA. Total RNA is denatured to expose the poly(A) (polyadenylated) tails. Poly(A)‐containing RNA is then bound to oligo(dT) cellulose, with the remainder of the RNA washing through. The poly(A)+ RNA is eluted by removing salt from the solution, thus destabilizing the dT:rA hybrid. The column can then be repeated to remove contaminating poly(A) RNA.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • Diethylpyrocarbonate (DEPC)
  • 5 M NaOH
  • Oligo(dT) cellulose
  • 0.1 M NaOH
  • recipePoly(A) loading buffer
  • 10 M LiCl
  • recipeMiddle wash buffer
  • 2 mM EDTA/0.1% sodium dodecyl sulfate (SDS)
  • 3 M sodium acetate
  • RNase‐free TE buffer
  • Silanized column ( appendix 3B)
  • Silanized SW‐55 centrifuge tubes ( appendix 3B)
  • Beckman SW‐55 rotor or equivalentThe following solutions should be treated with DEPC to inhibit RNase activity: water, 10 M LiCl, 3 M sodium acetate. See unit 4.1, reagents and solutions, for instructions.CAUTION:DEPC is a suspected carcinogen and should be handled carefully.
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Figures

Videos

Literature Cited

Literature Cited
   Aviv, H. and Leder, P. 1972. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid–cellulose. Proc. Natl. Acad. Sci. U.S.A. 69:1408‐1412.
   Moore, C.L. and Sharp, P.A. 1984. Site‐specific polyadenylation in a cell‐free reaction. Cell 36:581‐591.
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