Isolation of RNA from plant cell suspension cultures and calli by sonication.

Although several methods for RNA isolation from plants have been reported (3,4,7,10), there are no methods described for RNA isolation from plant cell suspension cultures and calli. During the characterization of transgene expression in tobacco cell suspension cultures and calli, we observed that it is difficult and tedious to isolate RNA from limited amounts of those cells frozen in liquid nitrogen by grinding in mortars with pestles because the wet cells harvested from suspension cultures or calli contain a lot of water. Therefore, we investigated alternative ways to isolate RNA from plant cells grown in culture. We describe a simple, reliable method for isolation of RNA from plant cell suspension cultures and calli by sonication that can be used to quickly process a large number of samples with either small or large quantities of plant materials. The plant materials used included nontransgenic tobacco (Nicotiana tabacum L.) BY-2 cell line (8), the transgenic BY-2 line transformed with the plant expression vector pSS carrying the single-chain antibody 24 sequence (scFv24) derived from a tobacco mosaic virus (TMV)-specific full-size antibody gene (9) and wheat (Triticum aestivum L.) cell suspension cultures generated from cultivar Prelude Sr5 (5). Two protocols used conventionally for isolation of RNA from plant leaves or other tissues by grinding were modified so that 0.2–5 g of plant suspension cultures or calli could be used for isolation of total RNA by sonication. Using the guanidinium protocol described by Wadsworth et al. (10), which is used here for small-scale preparation, 0.2 g (wet weight) of samples were mixed with 500 μL of Buffer A (4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 1.5% sodium lauroylsarcosine and 100 mM β-mercaptoethanol) in 2-mL microcentrifuge tubes. A 40T probe tip (B. Braun Biotech, Allentown, PA, USA) was held inside the tubes, and the samples were lysed by sonication on ice for 0.5–2 min (60 W, 0.5 s per cycle) or ground in the presence of liquid nitrogen in a mortar with a pestle. The lysed samples were subsequently processed following the protocol of Wadsworth et al. (10). The second protocol was based on the sodium dodecyl sulfate (SDS) method previously described for extraction of RNA from plant leaves (3), which is used here for large-scale preparations with minor modifications. Briefly, 5 g (wet weight) of samples were mixed with 12.5 mL of Buffer B (50 mM Tris-HCl, pH 9.0, 150 mM LiCl, 5 mM EDTA and 5% SDS) in 50-mL Falcon tubes (Becton Dickinson Labware, Bedford, MA, USA) and sonicated as described above. The samples were lysed by sonication on ice for 1–4 min (60 W, 0.5 s per cycle) or ground in the presence of liquid nitrogen with a mortar and pestle. The lysed samples were mixed with 1 vol of Buffer B and extracted twice with phenol/chloroform and once with chloroform/isoamyl alcohol. RNA in the aqueous phase was isolated as described in Reference 6. RNA concentrations were determined spectrophotometrically (1). The results indicated that the sonication method was very efficient for both tobacco cell suspension cultures and calli as well as wheat cell suspension cultures. Sonication resulted in average RNA yields ranging from 180 to 545 μg per gram (wet weight) (Table 1). The minimum sonication time is critical for efficient extraction of RNA. For tobac-