Late-stage IEs were isolated by magnetic separation as described (8) and returned to culture overnight to obtain ring-stage IEs. Genomic DNA was isolated with a QIAamp blood kit (Qiagen), and total RNA extracted (TRIzol, Invitrogen) and treated with DNase I (Invitrogen) for 30 min (10) . The absence of DNA in RNA samples was confirmed as described (11) . Reverse transcription was performed using Superscript II (Invitrogen) and random hexamer primers, followed by real-time PCR to quantify var transcript abundances as described (11) . We used primer pairs specific for the 59 var genes in the 3D7 genome (12) and for 45 var genes in the HB3 genome (Table S1 in Supporting Information). The latter were tested on genomic DNA dilutions to ascertain appropriate fragment size, melting temperature, and amplification efficiency compared to the internal control seryl-tRNA synthetase . All primer pairs varied less than two Ct values from that of the internal control and amplified single fragments of the expected sizes.