Protocol

Preparation of RNA from Plant Tissue Using Guanidinium Isothiocyanate/Cesium Chloride Ultracentrifugation

This protocol was adapted from "RNA Purification," contributed by Michael A. Connolly, Peter A. Clausen, and James G. Lazar, Chapter 10, in PCR Primer, 2nd edition (eds. Dieffenbach and Dveksler), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

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INTRODUCTION

This protocol is a modification of that outlined by Chirgwin et al. (1979). Tissues are homogenized in guanidinium isothiocyanate lysis buffer and then subjected to cesium chloride ultracentrifugation. The resulting RNA is then extracted with phenol and chloroform to remove protein and lipid contaminants.

MATERIALS

IMPORTANT: Prepare all reagents used in this protocol with DEPC-treated H₂O.

Reagents

Chloroform

CsCl/sodium acetate solution

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Equipment

METHOD