TABLE 1.

Chromosomal var gene transcription levels in various transformed parasite lines^a

FCB lines transformed by constructs	Ratio (mean ± SD) of transcript levels in parasite lines after normalization for:
	C341	C2143
pVDH, pVDH+intΔ2′	1.07 ± 0.89	0.81 ± 0.60
pVDH, pVDH+int149	2.74 ± 1.14	1.38 ± 0.45
pVDH, none	1.53 ± 0.33	2.45 ± 0.97
pVDH+intΔ2′, none	1.29 ± 0.04	1.63 ± 0.45
pVDH+int149, none	0.86 ± 0.19	2.32 ± 0.64

^aParasitized erythrocyte stages were separated on a six-step (40, 60, 70, 80, 85, and 90%) Percoll gradient in RPMI 1640-5% sorbitol by centrifugation at 12,000 × g for 20 min (room temperature). RNA was extracted from recovered parasites with a total RNA purification kit as recommended by the manufacturer (Invitrogen). Total RNA (1 to 3 μg) was treated with DNase for 15 min before RT (Invitrogen first-strand cDNA synthesis kit). Samples were primed with random hexamers. One sample in which reverse transcriptase was omitted served as a negative control to verify the absence of genomic DNA. Levels of transcription of var genes were determined by real-time quantitative RT-PCR with cDNA from transfected and nontransfected parasites and a Light Cycler (Roche Diagnostics, Indianapolis, Ind.). Five DNA standards containing 10⁸ to 10⁴ plasmid molecules were used for a standard curve. Amplification of the var cDNA DBLα domain with primers αAF [GCACG(A/C)AGTTTTGC] and αBR [GCCCATTC(G/C)TCGAACCA] (9) and amplification of C341 (disulfide-like protein) and C2143 (seryl tRNA synthetase) cDNA controls with the primers of Mamoun et al. (6) were performed in the same reaction tube. Five microliters of 10-fold-diluted cRNA or DNA standard was amplified in a 20-μl reaction mixture containing 9.6 μl of H₂O, 2.4 μl of MgCl₂ (25 mM), 0.5 μl of each primer (10 μM), and 2 μl of Fast Start DNA Master SYBR green I (Roche Diagnostics). PCR conditions were as follows: 10 min of denaturation at 95°C followed by 40 cycles of 95°C for 5 s, 48°C for 5 s, and 60°C for 25 s. Levels of expression from each transformed line were compared to each other and to that from untransformed FCB parasites and are presented as values normalized to those for C341 and C2143 control genes. All amplifications were repeated to ensure reproducibility (26 experiments for pVDH+intΔ2′, 24 experiments for pVDH+int149, and 15 experiments for untransformed FCB).