Genomic DNA and RNA Extraction for Malaria Parasites

Genomic DNA was extracted as described (18 (link)). Briefly, iRBCs from a 20 ml cultures were lysed with saponin and the parasite pellet was washed with PBS and taken up in 200 μl TSE buffer (100 mM NaCl, 50 mM EDTA, 20 mM Tris, pH 8), 40μl of 10% SDS and 20 μl 6M NaClO₄. This suspension was rocked for twelve hours and genomic DNA was extracted with phenol/chloroform. The resulting DNA was taken up in 100 μl dH₂O. RNA was extracted from synchronized parasite cultures at 20–24 h after percoll/sorbitol gradient centrifugation (16 (link),17 (link)). RNA was extracted with the TRIZOL LS Reagent^® as described (23 (link)) and purified on PureLink column (Invitrogen) according to manufacturer's protocol. Isolated RNA was then treated with DNase I (TaKaRa) to degrade contaminating gDNA. cDNA synthesis was performed from 500 ng total RNA with PrimeScript™ RT Reagent Kit (TaKaRa) as described by the manufacturer.