Revertaid h minus reverse

Quantification of liver-stage parasite burden was performed as described previously (Mol. Biochem. Parasitol. 2001, 118, p233–245). Briefly, at 30 h post-challenge, livers were perfused with PBS and removed. Total RNA was extracted with Trizol (Invitrogen, Darmstadt, Germany) according to the manufacturers instructions. RNA was transcribed using random hexamer primer and RevertAid H minus reverse transcriptase (Thermo Scientific, St. Leon-Rot, Germany) according to the manufacturers instructions. The resulting cDNA was amplified using the following primers: 5′-GGATGTATTCGCTTTATTTAATGCTT-3′ and 5′-CACGCGTGCAGCCTAGTAT-3′ for the detection of 18S rRNA of PbA. As reference gene mouse GAPDH was amplified with the primers 5′- GGGTGTGAACCACGAGAAAT-3′ and 5′-CCTTCCACAATGCCAAAGTT-3′. Cycling conditions were as following: 15 min 95°C, 40 cycles at 95°C for 15 s, 50°C for 20 s and 68°C for 20 s. For each cycle a melting curve analysis was performed with a ramp from 67° to 95°C. The relative concentration of P. berghei 18S rRNA was determined with the comparative C_t method (delta delta C_t).

The renal expression of fibrosis-related genes (TGFβ1 and ICAM1), matrix remodeling genes (ETS1, ITGβ2, and TIMP2), and the kidney injury-related gene (KIM1) following treatment with CM and/or its EXOs were detected by real-time PCR. First, total RNA was extracted from kidney specimens by using a Trizol reagent (Invitrogen, USA, Cat# 15596026). RNA concentration and purity were determined by using a Nanodrop (Q5000, Quawell, USA). Secondly, cDNA was synthesized from RNA by using the RevertAid H Minus Reverse (Thermo Scientific, #EP04 51). Finally, a PCR mix containing cDNA, 2XMaster Mix (QuantiTect SYBR Green), and primers (Table 1) was run in the Step One Plus thermal cycler (Applied Biosystem, USA) at the following conditions: one cycle initial denaturation at 94 °C for 4 min followed by 40 cycles of each denaturation at 94 °C for 40 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. The housekeeping gene β actin was utilized as an internal control. The results of qPCR were presented as fold change mean ± standard error of the mean (SEM) by using the Livak method (2^−∆∆Ct) and as previously described [50 (link)].

PV powder was purchased from Sigma Chemical Co. (St. Louis, MO, USA, Cat # P3510), CP (50 mg/50 mL) from EIMC United Pharmaceuticals (Cairo, Egypt), both Dulbecco’s Modified Eagle’s medium (DMEM) and heat-inactivated fetal bovine (FBS) serum from GIBCO (Grand Island, NY, USA), MTT from Molecular Probes (Eugene, OR, USA), dimethyl sulfoxide (DMSO) from Sigma Aldrich (Burlington, MA, USA), carboxymethylcellulose (CMC) from El-Gomhouria Company (Cairo, Egypt), Trizol reagent from Invitrogen (Waltham, MA, USA), RevertAid H Minus Reverse from Thermo Scientific (Waltham, MA, USA), and SYBR Green 2XMaster Mix from QuantiTect (Quiagen, Hilden, Germany). All other used chemicals were of high quality.