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Purification of Uncontaminated, Intact Plant RNA

  • Shu-Hua Cheng
  • Brandon D. Moore
  • Jeffrey R. Seemann
Protocol
  • 134 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The isolation of uncontaminated, intact RNA is essential for analyzing gene expression and for cloning genes. The presence of a large quantity of naturally occurring carbohydrates makes plant tissues one of the most difficult materials from which to isolate high-quality RNA with good yield. Such difficulty is due to copurification of carbohydrates during RNA isolation. These compounds form complexes with nucleic acids during tissue extraction and coprecipitate during subsequent alcohol precipitation steps (1, 2, 3, 4, 5). The resulting alcohol precipitates can be gelatinous and difficult to dissolve. An RNA solution contaminated with carbohydrates is viscous and absorbs strongly at 230 nm. This ultraviolet absorption prevents an accurate quantitation of RNA concentration by a measurement of A260 (see  Chapter 11). Furthermore, the contaminated RNA is not suitable for cDNA synthesis, reverse-transcription/polymerase chain reaction (RT-PCR) amplification, in vitro translation, or Northern blot analysis (6,7).

Keywords

Potassium Acetate Guanidinium Thiocyanate Longe Incubation Period Cesium Chloride Insoluble Polysaccharide 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Shu-Hua Cheng
    • 1
  • Brandon D. Moore
    • 2
  • Jeffrey R. Seemann
    • 2
  1. 1.Department of Molecular BiologyMassachusetts General HospitalBoston
  2. 2.Department of BiochemistryUniversity of NevadaReno

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