Purification of Uncontaminated, Intact Plant RNA
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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).
KeywordsPotassium Acetate Guanidinium Thiocyanate Longe Incubation Period Cesium Chloride Insoluble Polysaccharide
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