cDNA synthesis was performed using Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Invitrogen Canada, Burlington, ON, Canada) according to the manufacturer's protocol with minor modifications as follows. Five micrograms total RNA or 200 ng mRNA were used in each reaction. [32P]dGTP (Amersham Biosciences, Piscataway, NJ, USA) was used as tracer to monitor cDNA synthesis. After cDNA synthesis, reactions were purified from unincorporated nucleotides using a MicroSpin™ S300 column (Amersham Biosciences), and cDNA was separated on a denaturing 1% alkaline agarose gel. The gel was dried, exposed on a Phosphorimager Screen (Amersham Biosciences), and scanned on a Storm™ 860 phosphorimage scanner (Amersham Biosciences).Results and Discussion
The main objective of this study was to develop one method that works reliably for RNA isolation from different woody plants, including angiosperms and gymnosperms, for subsequent application in tree genomics (e.g., reverse transcription of RNA for EST and full-length cDNA library construction and for microarray RNA expression profiling). The protocol described here was specifically optimized for use in comparative tree genome research, which requires large numbers of isolations of consistently high-quality RNA from diverse species and tissues.
Several existing methods deal with different problems of RNA isolation from conifers. A widely used method described by Chang et al. (1), as well as a number of recent protocols (3,4), use preheated CTAB and extraction buffers with high concentrations of NaCl to remove high levels of polysaccharides and proteins during RNA isolation. The method described in Reference 1, was developed for RNA isolation from conifers, and in our hands, it yielded RNA of good quality when applied to conifer tissues. However, other reports (5,6) and our test of this method with poplar tissues revealed some limitations of the method. Lewinsohn et al. (5) could not obtain intact RNA from conifers using this protocol, and Wu et al. (6) describe similar problems when isolating RNA from cotton. When we extracted RNA from poplar leaves using the protocol described in Reference 1, the RNA quality was inconsistent. In several instances, use of the method reported in Reference 1, produced largely degraded RNA, probably due to the presence of active RNases during incubation at 65°C right after tissue homogenization.
Methods described by Lewinsohn et al. (5) and Wang et al. (2) efficiently remove excessive phenolics with PVPP in the extraction buffer. However, in our experiments, the protocol described in Reference 2, yielded relatively low amounts of RNA from spruce needles and spruce xylem (Table 1), and RNA from spruce xylem and all poplar tissues tested sometimes contained large amounts of gelatinous polysaccharides. Polysaccharides co-precipitate with RNA (as mentioned in Reference 5) and do not separate from RNA in later steps of mRNA isolation. The resulting mRNA was not suitable on a reliable basis for reverse transcription reactions and subsequent cDNA library construction. Another RNA isolation method, described by Mattheus et al. (7), requires time-consuming cesium chloride ultracentrifugation and was therefore not taken into consideration for application for large sample numbers typical of genomics research. In conclusion, none of the existing methods tested in our initial trials fulfilled the requirements for a protocol that can be widely used in tree genomics research on a large sample number with both gymnosperms and angiosperms.
By combining key features of the methods described by Chang et al. (1) and Wang et al. (2), we developed an improved RNA isolation protocol that was suitable for RNA isolation from conifer and poplar tissues rich in oleoresins, phenolics, or polysaccharides. Using this method, we consistently obtained high yields of intact RNA (Table 1). We adopted the extraction buffer described by Wang et al. (2) because it inactivates polyphenolics, includes RNase inhibitors, and does not require incubation of tissue extracts at elevated temperatures prior to removal of RNases. After removal of RNases, we introduced a polysaccharide purification step using the CTAB method described in Reference 1,. Incubation at elevated temperatures was kept to a minimum.