Trizol tri reagent

A 10 mL culture of Acinetobacter sp. cells growing in either LB or Minimal Media, supplemented or not with 1mM InsP₆ or inorganic phosphate (0.365 mM), equivalent to that impurity in the InsP₆, was extracted after reaching late exponential phase. Cells were centrifuged at 6000 x RPM for 5 minutes and to the pellet 1 mL of TRIzol/TRI reagent (Invitrogen) was added. Tubes were vortexed and incubated at room temperature for 5 minutes. Chloroform (0.2mL) was added, the tubes vortexed, incubated at room temperature for 2 minutes and centrifuged at 13,000 x RPM for 10 minutes at 4°C. Isopropanol (500 μL) was added to the (removed) upper aqueous phase, mixed by inversion and incubated for 10 minutes at room temperature to precipitate RNA. Pelleted (12,000 x RPM, 10 minutes, 4°C) RNA was washed with 1 mL of 75% ethanol, centrifuged, air-dried, and resuspended in 30 μL RNase-free water.

A total of 112 samples were collected from patients aged 6 months–13 years, who visited the health centre with uncomplicated malaria from June to August 2014. Children who were found to be P. falciparum positive by microscopy were enrolled after parental consent was obtained and in accordance with the study inclusion criteria [26 ]. Sixteen of the children did not return for the 1 week follow up visit. Prior to treatment (day 0) and during the 7 day follow up visit (day 7), 2 ml of venous blood was collected into EDTA vacutainer tubes and an aliquot spotted onto filter paper (Whatman^® 3 mm). The filter paper was air-dried and stored desiccated at room temperature. The EDTA samples were immediately centrifuged and the plasma collected and saved for future use. One hundred microlitres of pelleted cells were preserved in 500 µl of Trizol (Tri Reagent, Invitrogen). All the samples were transported to the NMIMR for analysis.