Accugene

Filter paper samples were air dried in the field, put in sealable bags with silica and transported the same day to the laboratory at the Clinical Research Unit of Nanoro (CRUN, Burkina Faso). The dried blood spots on filter papers were kept at ambient temperature until shipment to the Institute of Tropical Medicine (ITM, Belgium) for processing. Genomic DNA was extracted from 3 punches of dried blood spots (5 mm in diameter) using QIAamp 96 DNA blood kit (Qiagen, Germany), following the manufacturer’s recommendations and a final elution in 150 µL of water (Lonza, AccuGENE). Five µL of DNA were used as template for qPCR analysis targeting P. falciparum var gene acidic terminal sequence (varATS, ≈59 copies per genome) as previously described [38 (link)]. Parasite densities were obtained by interpolating cycle thresholds (Ct) using a standard curve prepared with titrated samples containing known numbers of infected erythrocytes diluted in whole blood (100,000–0.01 parasites/μL). The limit of detection of the varATS-based qPCR was 0.1 parasite/μL. Samples with Ct value > 39.7 were considered negative. DNA extracted from P. falciparum obtained from reference 3D7 parasites cultures was used as positive control. The negative controls included human negative blood spots on filter paper and mastermix reagent used as no template control (NTC).

Fresh cell pellets or lyophilized powders containing 3 million cells were carefully resuspended and vortexed for 5 min in 1 ml TRIzolate reagent (UD-Genomed Medical Genomic Technologies Ltd., cat. URN0103). Phase separation was carried out using chloroform (1:5) (Sigma-Aldrich, cat. C2432) and high-speed centrifugation. RNA was precipitated from the aqueous phase for 10 min at room temperature using isopropanol (1:1) (Sigma-Aldrich, cat. I9516). Pellets were washed twice with chilled 75% ethanol (diluted with nuclease-free water from absolute ethanol, VWR International, cat. 20821.296), vacuum-concentrated and redissolved in nuclease-free water (AccuGENE, Lonza, cat. 51200) at 65° C for 10 min. Sample purity was determined using a NanoDrop 1000 instrument (Thermo Fisher Scientific, Waltham, MA, USA), and accurate concentrations were determined using a Qubit RNA HS Assay Kit (Thermo Fisher Scientific, cat. Q32855). Each RNA sample was loaded on Agilent RNA 6000 Nano microchips (Agilent, Santa Clara, CA, USA) according to the manufacturer’s recommendations for the analysis of total RNA fragment distribution and calculation of RIN values.

Single-strained DNA probe for miRNA-16b target recognition was purchased from Integrated DNA Technologies, Inc. (IDT, Coralville, IA). Its sequence was 5′- GCCAATATTTACGTGCTGCTGCTA-3′ while the 5′-end was dithiol-modified for self-assembly on the gold surface. Before experiments, the ssDNA probes were further reduced using TCEP (Sigma Aldrich, USA). The miRNA-16b was purchased from IDT and its sequence was rUrArG rCrArG rCrArC rGrUrA rArArU rArUrU rGrGrC [10 (link)]. For the sensor selectivity test, miRNA-25 [11 (link)], also from IDT, had the following sequence: rCrArU rUrGrC rArCrU rUrGrU rCrUrC rGrGrU rCrUrG. After DNA probe incubation, the surface of the electrode was blocked with 1 mM 6-MH (Sigma-Aldrich, St. Louis, MO, USA). For probe solution, ultrapure water (Mili-Q) and 0.05× PBS (Thermo Fisher Scientific, Waltham, MA, USA) were used to prepare 20 µM DNA probe solution. For testing, miRNA-16 was diluted in 1× SSC buffer (AccuGENE™, Lonza, Rockland, ME) to make stock solutions. The final concentrations of miRNA-16b samples for testing were: 100 fM, 10 fM, 1 fM and 0.1 fM.