Abstract
BackgroundHigh quality RNA is a primary requisite for numerous molecular biological applications but is difficult to isolate from several plants rich in polysaccharides, polyphenolics and other secondary metabolites. These compounds either bind with nucleic acids or often co-precipitate at the final step and many times cannot be removed by conventional methods and kits. Addition of vinyl-pyrollidone polymers in extraction buffer efficiently removes polyphenolics to some extent, but, it failed in case of Azadirachta indica and several other medicinal and aromatic plants.FindingsHere we report the use of adsorption property of activated charcoal (0.03%–0.1%) in RNA isolation procedures to remove complex secondary metabolites and polyphenolics to yield good quality RNA from Azadirachta indica. We tested and validated our modified RNA isolation method across 21 different plants including Andrographis paniculata, Aloe vera, Rosa damascena, Pelargonium graveolens, Phyllanthus amarus etc. from 13 other different families, many of which are considered as tough system for isolating RNA. The A260/280 ratio of the extracted RNA ranged between 1.8-2.0 and distinct 28S and 18S ribosomal RNA bands were observed in denaturing agarose gel electrophoresis. Analysis using Agilent 2100 Bioanalyzer revealed intact total RNA yield with very good RNA Integrity Number.ConclusionsThe RNA isolated by our modified method was found to be of high quality and amenable for sensitive downstream molecular applications like subtractive library construction and RT-PCR. This modified RNA isolation procedure would aid and accelerate the biotechnological studies in complex medicinal and aromatic plants which are extremely rich in secondary metabolic compounds.
Highlights
High quality RNA is a primary requisite for numerous molecular biological applications but is difficult to isolate from several plants rich in polysaccharides, polyphenolics and other secondary metabolites
We attempted the use of Polyvinyl pyrrolidone (PVP) or/and Polyvinyl polypyrrolidone (PVPP) in the guanidine ion-based extraction buffers which makes complexes with polyphenolics through H-bonding, but could not get any RNA yield
We believed that the failures in the RNA extraction from A. indica tissues by using standard procedures were because of the fact that the plant is highly rich in polyphenolics and other complex secondary metabolites
Summary
High quality RNA is a primary requisite for numerous molecular biological applications but is difficult to isolate from several plants rich in polysaccharides, polyphenolics and other secondary metabolites. Since standard RNA extraction methods like guanidine thiocyanate phenolchloroform method [4], modified hot borate method [5], and cetyltrimethylammonium bromide (CTAB) method [6] failed to yield appropriate quality and quantity of RNA from several plants rich in secondary metabolites and polysaccharides, modifications in the extraction procedures were attempted by several researchers to isolate good quality RNA for downstream applications Their countermeasures for the removal of polyphenolics, polysaccharides and other complex secondary metabolites were the increase in the volume of extraction buffer in order to dilute the polyphenolics and the addition of vinyl-pyrollidone polymers (PVP or PVPP) in the extraction buffer [7,8]. Extraction method using water saturated phenol followed by acetic acid precipitation keeps the pH low (acidic) which increases the RNA stability [11], and, employing extraction buffers having high ionic strength in combination with lithium chloride precipitation effectively prevents the co-precipitation of polysaccharides along with nucleic acids [12] These modifications in RNA extraction procedures failed in case of diverse plant systems having complex secondary compounds. It has been successfully used in plant tissue culture to prevent growth inhibition due to excess polyphenolics which are released into the medium [17] as well as to remove PCR inhibitors and increase the sensitivity of real-time PCR reactions [18]
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