Geneq thermal cycler

Total RNA (1 µg) of E. histolytica trophozoites cultured under different conditions as described above was obtained by the TRIzol reagent (Invitrogen) according to manufacturer instructions and used to validate PPI networks predictions and RNA seq datasets analysis through Real-Time qRT-PCR. For this, cDNA was synthesized using the SuperScript III Reverse Transcriptase (Invitrogen) according to manufacturer’s instructions in a GeneQ Thermal Cycler (BIOER, Hangzhou, China). The qPCR assay was completed using the SensiFAST™ SYBR Hi-ROX Kit (Bioline) with specific primers for selected genes (Table 1). All reactions were performed in a StepOne real-time PCR system (Applied Biosystems) with the following steps: enzyme activation at 95°C for 2 min, denaturation at 95°C for 5 s, and annealing/extension at 60°C for 30 s (40 cycles). Experiments were performed by triplicate with three biological samples and the relative expression of mRNA was determined by the 2^−ΔΔCt method using data of the EhRNAPII gene for normalization [44 (link)].

The relative expression levels of genes of interest were studied using RT-PCR, and total RNA for this purpose was extracted using the GenElute™ Mammalian Total RNA Miniprep kit (Sigma-Aldrich). RNA concentration and purity were determined spectrophotometrically, and equal amounts of RNA were reverse transcribed into cDNA using M-MLV reverse transcriptase (Top-Bio, Prague, Czech Republic). RT-PCR was carried out in 20-μL reaction volumes using the GeneQ thermal cycler (BIOER). To ensure that the amplification does not reach a saturation plateau, the suitable number of cycles for analysis of each gene in question was selected on the basis of comparison of RT-PCR products after 25, 30, 35 and 40 cycles of reaction (S1 Fig). According to this initial analysis, expression level of each gene was semi-quantified after the 30 cycles of reaction, i.e. in the exponential phase of amplification. The products were subsequently separated with 1% agarose gel electrophoresis and visualized in a UV transilluminator (UVITEC Cambridge). Visualized transcript bands were quantified using ImageJ. The reference genes HSP90AB1 and GAPDH were used as an endogenous control. Data were analyzed using one-sample T-test (two-tailed); p< 0.05 was considered statistically significant. The primers used in this study are listed in Table 2.