Comparison of Methods for Isolating High-Quality RNA from Leaves of Grapevine

Am. J. Enol. Vitic. 56:4 (2005) 400 Extraction of high-quality total RNA is essential for the successful application of many molecular techniques, such as reverse transcription polymerase chain reaction (RTPCR), cDNA library construction, and gene expression profiling studies using microarrays. RNA is degraded rapidly by ribonucleases (RNases) and, therefore, must be extracted quickly and efficiently (Sambrook et al. 1989). Denaturing reagents such as phenol (Davies and Robinson 1996) or guanidine thiocyanate (John 1992, Salzman et al. 1999) and inhibitors of RNases such as aurintricarboxylic acid (ATA) (Franke et al. 1995, Lewinsohn et al. 1994) are often added to extraction buffers. Extraction of high-quality RNA from the leaves of woody plants, such as grapevine, is particularly challenging because of high concentrations of polysaccharides, polyphenols, and other secondary metabolites (Loulakakis et al. 1996, Salzman et al. 1999). When applied to grape leaves, some RNA extraction methods yield pellets that are poorly soluble, indicating the presence of unknown contaminants, whereas others are gelatinous, indicating the presence of polysaccharides (Tesniere and Vayda 1991). Other procedures yield reddish pellets indicative of phenolic contamination (Bahloul and Burkard 1993, Katterman and Shattuck 1983). RNA can become complexed with polysaccharides and phenolic compounds (Bahloul and Burkard 1993, Lewinsohn et al. 1994, Salzman et al. 1999), particularly if chaotropic agents (Newbury and Possingham 1977) or vinyl-pyrrolidone polymers and antioxidants (Salzman et al. 1999) are not present in the extraction buffer. Polysaccharide and phenolic complexes render the RNA unusable for applications such as reverse transcription and cDNA library construction (Salzman et al. 1999). Whereas some standard procedures developed for model plant species, such as Arabidopsis and rice (for example, TRIzol, RNAwiz, RNeasy Plant), are very reliable, these methods are often inadequate when applied to leaves of Vitis vinifera. While several reports using recalcitrant plant tissues compared a few methods of RNA isolation (Logemann et al. 1987, Loulakakis et al. 1996, Tesniere and Vayda 1991), the scope of these comparisons was relatively limited. Logemann et al. (1987) compared six methods using various combinations of cetyltrimethylTechnical Brief Comparison of Methods for Isolating High-Quality RNA from Leaves of Grapevine