High-quality RNA Isolation from Pigment-rich Flowers
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Isolation of high-quality RNA from flowers is challenging because of the high levels of pigment, polysaccharides, and polyphenols. In the present study, an efficient CTAB method for RNA extraction from the pigment-rich flowers of was optimised. The optimised method yielded high quantities of RNA (10.1-12.9 µg/g). Spectrophotometric values of A in the range of 2.2 to 2.4 and A values of 2.0 suggested that the isolated RNA was free of polyphenols, polysaccharides, and protein contaminants. RNA integrity numbers determined by microfluidics were in the range of 7.9-8.9 indicative of intact RNA. In the improved method, the addition of 3 M NaCl and 3% PVP-10 in the extraction buffer, followed by an incubation period of 45 min at 65 °C, eliminated most of the polysaccharides, polyphenolic compounds, and denatured protein. Extraction with phenol:chloroform:isoamyl alcohol (125:24:1) effectively removed pigments from the aqueous phase, while the precipitation of RNA with lithium chloride minimised the co-precipitation of protein, DNA, and polysaccharide and resulted in the extraction of high quality of RNA. The suitability of the RNA for downstream processing was confirmed via RT-PCR amplification of gene from cDNA prepared from RNA isolated from different developmental stages of the flower of a hybrid. The present method will be highly useful for the isolation of RNA from pigment, polyphenol, and polysaccharide-rich plant tissues.
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