Simultaneous extraction of DNA and RNA from nuclease-rich pathogenic protozoan Trichomonas vaginalis.

Trichomoniasis, caused by the colonization of Trichomonas vaginalis in the urogenital tract, is one of the most prevalent sexually transmitted human diseases. Efforts to extract high-quality nucleic acids for studies have often been hampered by the potent nuclease activities released upon lysis of T. vaginalis (1–3,6,7). Protocols to isolate DNA from T. vaginalis, such as the guanidinium-HCl method (7) and the diethyl pyrocarbonate (DEPC)-Triton X-100 method (2), are often laborious, time-consuming and technically demanding. They also require a large cell mass with which to work, and the yields are always unsatisfactory and inconsistent. Here we report a rapid protocol to simultaneously extract DNA and RNA from T. vaginalis. T. vaginalis T1 isolate (5) was maintained in an axenic culture as described elsewhere (7). The cells were harvested from 13 mL of the logarithmic phase culture by centrifugation at 900× g for 10 min in a table-top centrifuge. The cells (ca. 2 × 107) were transferred to a 1.5-mL Eppendorf tube (Eppendorf North America, Madison, WI, USA) and washed with 1 mL of sterile phosphate-buffered saline (PBS). The cell mass was collected by centrifuging in a microcentrifuge for 1 min at 4°C, and then resuspended in 50 μL of lysis buffer containing 1% Triton X-100 in sterile PBS. The suspension was vigorously agitated by vortex mixing for 30 s immediately before spinning in a microcentrifuge for 1 min at 4°C. The Triton-soluble fraction was transferred to a fresh Eppendorf tube, and the Tritoninsoluble fraction was resuspended in 50 μL lysis buffer. Sodium dodecyl sulfate (SDS) was added to both fractions to give a final concentration of 1%, and the nucleic acids were immediately extracted with water-saturated phenol (pH 4.3) at 65°C as described (4). Subsequently, 10 μL of the nucleic acids in each fraction were examined by electrophoresis in a 1% agarose gel. Cellular RNA and a 4.6-kb viral genomic double-stranded (ds) RNA of T. vaginalis virus (5) were found in the Tritonsoluble fraction (Figure 1, lane 1), while the high molecular weight DNA molecules remained in the Triton-insoluble fraction (Figure 1, lane 2). RNA in the Triton-soluble fraction was extracted once more with water-saturated phenol (pH 4.3) at 65°C to remove active residual nucleases. The purified RNA was suitable for the biosynthesis of first-strand cDNA using reverse transcriptase and subsequent amplification by polymerase chain reaction (PCR) (data not shown). RNA was removed from the Triton-insoluble fraction by RNase digestion to obtain pure DNA. The nucleic acids were further precipitated with ethanol. Approximately 8 μg of the DNA were obtained. Incubation of the purified DNA at 37°C for 20 h did not cause significant degradation as compared to the unincubated sample (data not shown). DNA isolated by this method is compatible with restriction enzyme digestion (Figure 2). An enzyme-specific digestion pattern was observed, indicating the existence of repetitive sequences in the genomic DNA of T. vaginalis. DNA purified by this method was suitable for PCR amplification and molecular cloning (data not shown). It is stable for several months when stored at -20°C (data not shown). In contrast, DNA prepared by the DEPC-Triton X-100 method (2) and stored under similar conditions in our laboratory was degraded (data not shown). This method can be scaled up in a single 1.5-mL Eppendorf tube to obtain high-quality DNA (ca. 60 μg) from 2 × 108 of the cells (resuspended in 500 μL lysis buffer). A much larger quantity of DNA (ca. 1 mg) was obtained by further scaling up the extraction in a 30-mL Corex tube (Corning Costar, Cambridge, MA, USA); however, RNA was degraded in this procedure. Development of a rapid method of extracting nucleic acids is essential to overcome the nuclease activities released upon lysis of T. vaginalis. The method described above should be performed as quickly as possible or RNA will be degraded. The quality of DNA