SmPK, SmPLP-Ph, and SmPNPO Gene Expression Analysis

The levels of expression of the SmPK, SmPLP-Ph and SmPNPO genes in different life stages of the parasite was measured by reverse transcription quantitative PCR (RT-qPCR), using a custom TaqMan gene expression system (Applied Biosystems). RNA was isolated from different stages of the parasites using TRIzol reagent and cDNA was synthesized as described above. The levels of expression of these genes in different life stages was measured by RT-qPCR using the housekeeping gene triose phosphate isomerase as the endogenous control (27). Primer sets and reporter probes labeled with 6-carboxyfluorescein (FAM), obtained from Applied Biosystems were used. To monitor gene expression, the following primers and probe were used: SmPK-F: 5’-TTGAAAATGAAAACGGACAACAAATAGCT-3’, SmPK-R: 5’-GATAGAACAGTTTGGATTGTACTAAGAACTGA-3’, SmPK probe: 5’-FAM-TTCCTTCAAAGATGCCTTCC-3’; SmPLP-Ph-F: 5’-GTTGCGAATTGGACCCTTCAAAA-3’, SmPLP-Ph-R: 5’-CCAAATTTATTCCCAAATGCGATATCAGT-3’, SmPLP-Ph-probe: 5’-FAM-CTGTGATGGTTGGAGATAACTTAT-3’; SmPNPO-F: 5’-GAAAATGAATATGCTTCAAAAGAAAAGCTTCC-3’, SmPNPO-R: 5’-CCAAAACTCAATTGATTTCGGAGACA-3’, SmPNPO-probe: 5’FAM-TCCCCAATTTGAAGGCTTT-3’. Each RT-qPCR was performed using a TaqMan PCR mix, cDNA and primer and probes in a final volume of 20 µl. All samples were run in triplicate and underwent 40 amplification cycles on a Step One Plus Real Time PCR System Instrument. For relative quantification, the ΔΔCt method was employed.