FIG. 1.

RNA isolation and PCR analysis of subtraction efficiency. (A) Total RNA was isolated from primed parasites after contact with VECs (pTv) and control organisms handled identically (T. vaginalis [Tv]). Total RNA (3 μg) was separated on a 1.2% agarose gel followed by staining with EtBr to visualize the purity and assess degradation of RNA. 28S and 18S refer to the rRNA bands of the VECs and the parasites. (B) PCR using α-tubulin primers was performed on unsubtracted and subtracted cDNAs. Aliquots (5 μl) were removed at a predetermined number of cycles and analyzed by EtBr-stained gels after electrophoresis in 1.2% agarose. The α-tubulin product appeared only after 25 cycles in the subtracted sample compared to 15 cycles in the unsubtracted sample.