Isolation of good quality RNA from a medicinal plant seabuckthorn, rich in secondary metabolites

Medicinal plants are being widely investigated owing to their ability to produce molecules of therapeutic significance. Isolation of good quality RNA is a tedious but primary step towards undertaking molecular biology experiments. However, medicinal plants are rich in secondary metabolites and not amenable to standard RNA isolation protocols involving Guanidine isothiocyanate (GITC). So an RNA isolation protocol from difficult samples (richer in secondary metabolites) is of highest desiderata. Here we propose a new protocol suitable for isolating RNA from plant tissues rich in secondary metabolites. To standard CTAB (Cetyl Trimethyl Ammonium Bromide) buffer, addition of 2% PVPP (polyvinyl polypyrrolidone) and 350 mM beta-mercaptoethanol was found useful. Use of glacial acetic acid (1M) along with ethanol for precipitation after phenolization and chloroform extraction enhanced the RNA yield. This is the first report of using glacial acetic acid in a CTAB based protocol for the precipitation of RNA. This protocol has been validated in medicinal plant Hippophae rhamnoides vern. seabuckthorn, where standard RNA isolation methods involving GITC and TRIZol extraction buffers failed. The RNA isolated by this method was of good quality as gauged by spectrophotometric readings and denaturing agarose gel electrophoresis. To the best of our knowledge, this RNA isolation protocol has never been published before. The RNA thus obtained could be suitably used for the downstream molecular procedures like Reverse Transcription Polymerase Chain Reaction (RT-PCR), Real Time-PCR, cDNA library construction, etc. (C) 2009 Elsevier Masson SAS. All rights reserved.