Preparation of Yeast RNA

Martine A. Collart1, Salvatore Oliviero1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 13.12
DOI:  10.1002/0471142727.mb1312s23
Online Posting Date:  May, 2001
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Abstract

This unit provides two protocols for extraction of RNA from yeast that differ primarily in the method for lysing the yeast cells. The first protocol isolates RNA directly from intact yeast cells by extraction with hot acidic phenol. This yields RNA that is relatively free of contaminating DNA, is convenient to perform with multiple samples, and gives little or no sample‐to‐sample variation. In contrast, an alternate protocol relies upon disruption of cells by vigorous mixing with glass beads and denaturing agents. Although this procedure results in efficient breaking of the cells, the product is associated with residual DNA, and the procedure itself is troublesome when one is working with multiple samples. A second alternate protocol describes the scaling up of the first two procedures to isolate enough total RNA for poly (A)+ RNA preparation.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Yeast RNA by Extraction with Hot Acidic Phenol
  • Alternate Protocol 1: Preparation of RNA Using Glass Beads
  • Alternate Protocol 2: Preparation of Poly(A)+RNA
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Preparation of Yeast RNA by Extraction with Hot Acidic Phenol

  Materials
  • Yeast cells and desired medium (units 13.1 & 13.2)
  • recipeTES solution
  • recipeAcid phenol
  • Chloroform
  • 3 M sodium acetate, pH 5.3
  • 100% and 70% ethanol, ice‐cold
  • 50‐ml centrifuge tube (Falcon)
  • Centrifuge: tabletop or Sorvall equipped with an SS‐34 rotor
  • Additional reagents and equipment for ethanol precipitation (unit 2.1) and spectrophotometric quantitation of cells and RNA ( appendix 33)

Alternate Protocol 1: Preparation of RNA Using Glass Beads

  Additional Materials
  • recipeRNA buffer
  • 25:24:1 phenol/chloroform/isoamyl alcohol (equilibrated with RNA buffer; see support protocol, unit 2.1)
  • 0.45‐ to 0.55‐mm, chilled, acid‐washed glass beads (Sigma)
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Literature Cited

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