Isolation and Purification of RNA from Tissues Rich in Polyphenols, Polysaccharides, and Pigments of Annatto (Bixa orellana L.)
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The tropical plant Bixa orellana L. (annatto) produces an array of natural products, including the pigment bixin used in the food and cosmetics industries. In order to understand the biochemical and molecular basis of the biosynthesis of these natural products, a reliable method for isolating high yields of high-quality RNA is required. Here we described a successful and reproducible method for isolation and purification of high-quantity and high-quality RNA from different tissues of annatto. This protocol overcomes the usual problems associated with large amounts of polyphenols, polysaccharides, pigments, and other secondary metabolites that are not easily removed by conventional extraction procedures. Furthermore, the proposed protocol can be easily carried out in any laboratory and it could also be extended to isolate RNA from other plant species showing similar abundance of compounds that interfere with RNA extractions. The yield and quality of the RNA were monitored by spectrophotometric analysis, separation on agarose gel, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and construction of a cDNA library.
KeywordsRNA extraction MATAB (mixed alkyl trimethyl ammonium bromide) RT-PCR cDNA library
This research was supported by CNPq (Research grant no. 473619/04-04 to WCO) and BNB (Research grant no. 2004-1-22 to MGCC). We gratefully acknowledge the Ph.D. scholarships to S. M. Rodrigues and V. L. F. Soares by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB), respectively. We thank F. T. S. Nogueira (Cold Spring Harbor Laboratory, USA) for the critical reading of the manuscript. T. M. de Oliveira is recipient of a PIBIC fellowship from CNPq.
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