Isolation and Purification of RNA from Tissues Rich in Polyphenols, Polysaccharides, and Pigments of Annatto (Bixa orellana L.)

Abstract

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.

Methodology An alternative cetyltrimethylammonium bromide-based protocol for RNA isolation from blackberry (Rubus L.)

“…Other conventional methods employing the hash lysate buffer such as sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), N-lauroyl sarcosine, and urea have also been widely used for RNA isolation in plant species (Liao et al, 2004;Almarza et al, 2006;Fort et al, 2008;Rio et al, 2010;Wu et al, 2011). Special measures could be adopted to address the problems caused by inferences of polysaccharides, phenolic compounds, starches, and pigments (Rodrigues et al, 2007;Kansal et al, 2008;Smart and Roden, 2010).…”

Isolation of retro-transcribed RNA from in vitro Mycosphaerella fijiensis-infected banana leaves

“…Our research required the isolation of high-quality RNA from in vitro banana plantlet leaves, which has proved to be an ongoing challenge. Initially, we compared the Trizol ® reagent and CTAB or SDS-containing protocols, also we tested the method reported by Rodrigues et al (2007), but even after repeated adjustments, these produced low yields or poor-quality RNA (data not shown). We then tested three protocols commonly used to eliminate polyphenols, polysaccharides and proteins: Concert TM Plant RNA Reagent, a commercially available total RNA extraction kit appropriate for eliminating these metabolites from plant tissues; a small-scale protocol developed to extract RNA from cactus fruit rich in polysaccharides (Valderrama-Cháirez et al, 2002), which has been successfully applied with Tagetes erecta and prickly pear (Rosas-Cárdenas et al, 2007), and a modified version of this protocol.…”

Efficient Strategies for Elimination of Phenolic Compounds During DNA Extraction from Roots of Pistacia vera L.

“…The present protocol could successfully extract DNA from samples however, the aim of this study was not focusing on the yield of DNA, in fact the main objective was removing polyphenolic compounds which precipitated concomitant with DNA through nucleic acid extraction. Polyphenolic contamination of DNA was determined by A260/A230 ratio, the ratio closed to 2 or > 2 showed a very low or no contamination in DNA (Kasem, Rice, & Henry, 2008;Rodrigues et al, 2007). The A260/A230 ratio of extracted DNAs by introduced method varied between 1.55-1.80 whereas the ratio for extracted DNAs using commercial kit was very low between 0.02-0.25, indicating the presence of organic contaminants (Table 1).…”