Regulators of sexual commitment in Plasmodium falciparum

The aim of this study was to identify transcriptonal responses during sexual commitment of P. falciparum mutants unable to form gametocytes. Sexual induction of the kinase KO lines and 3D7 control line was started with a 3% asexual ring culture (cycle I) where sexual commitment was induced by using 50% spent medium during the following intra-erythrocytic cycle (cycle II). The sexually committed merozoites were expected to invade and gametocytes develop during the next cycle of infection (cycle III). The samples were collected at 28-32 hpi during cycle II or induction cycle (Time Point I) and at 28-32 hpi after induction   of sexual commitment during cycle III (Time Point II). The infected RBCs were collected at the respective time point, centrifuged and solubilized in ten volumes of TRIzol prewarmed to 37°C, mixed vigorously for 5 minutes at 37°C and immediately frozen at -80°C until extraction. Once all samples were collected, the frozen samples were quickly thawed and 0.2mL of chloroform added per each mL of TRIzol. After vigorous mixing the mixture was left for 3 min at room temperature. After centrifugation at 13,200 rpm for 30min at 4°C, the upper aqueous phase containing the RNA was collected into a new tube and an equal volume of ethanol 70% added. After vigorous mixing, the solution was loaded into an RNA Spin cartridge purified using the RNAeasy Quiagen. After RNA extraction and purification, the samples were treated with DNAse I (Quiagen). The synthesis of cDNA was performed using the superscript III first-strand synthesis system (Invitrogen). Only samples collected at time point I (Induction cycle) were considered for the purpose of the paper.