title: Activated charcoal-mediated RNA extraction method for Azadirachta indica and plants highly rich in polyphenolics, polysaccharides and other complex secondary compounds.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  Here 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India.
authors.forename: Raja
authors.initials: R
authors.lastname: Rajakani
authors.forename: Lokesh
authors.initials: L
authors.lastname: Narnoliya
authors.forename: Neelam Singh
authors.initials: NS
authors.lastname: Sangwan
authors.forename: Rajender Singh
authors.initials: RS
authors.lastname: Sangwan
authors.forename: Vikrant
authors.initials: V
authors.lastname: Gupta
mesh_terms: D031601:Azadirachta / Q000737:chemistry*; D002606:Charcoal / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D020224:Expressed Sequence Tags; D015723:Gene Library; D009693:Nucleic Acid Hybridization; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology*; D059808:Polyphenols / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D018749:RNA, Plant / Q000032:analysis / Q000302:isolation & purification*; D012335:RNA, Ribosomal / Q000032:analysis / Q000302:isolation & purification*
chemical_list: D010936:Plant Extracts; D059808:Polyphenols; D011134:Polysaccharides; D018749:RNA, Plant; D012335:RNA, Ribosomal; D002606:Charcoal
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