Isolation and purification of functional total RNA from woody branches andneedles of Sitka and white spruce

The isolation of intact, functional RNA from conifer spp. is not easy, espe cially from those tissues that are heavily lignified and characterized by a low number of living cells. An efficient procedure for isolating RNA from combined wood and bark tissues of conifers was developed based on a protoco l optimized for the extraction of RNA from pollen and one for the isolation of RNA from woody stems. This protocol does not involve the use of phenol, and no ultracentrifugation was required In addition, the protocol overcame the problems of RNA degradation and low yield due to oxidation by polyphen olics and co-precipitation with polysaccharides, both of which are abundant components in conifer bark tissues. The isolated RNA was of high quality a nd undegraded as gauged by spectrophotometric readings and electrophoresis in denaturing agarose gals. Quality was further assessed through the subseq uent use of the RNA in reverse transcription and RT-PCR, indicating that it could be used for a number of downstream purposes including Northern blot hybridization and cDNA library construction. Using this modified protocol; 80-150 mu g of RNA was routinely obtained from 1 g of flesh material. This protocol was also used for the isolation of RNA from needles of spruce spp. , from which 750-950 mu g RNA per gram of starting material could routinely be obtained.