Platinum sybr green qpcr supermix udg with rox reagent

For the CK and CKR genes, a standard curve from serial dilutions of a known concentration of purified DNA was achieved. This quantified DNA consists of the target PCR product prepared by conventional PCR, from cDNA positive for the corresponding target mRNA. Threefold measurements, for each standard dilution point over the whole standard curve range, produced to generate a reliable standard curve. Then, real-time PCR quantitative mRNA analyses were performed using an ABI Prism 7000 SDS unit (Applied Biosystems) through the Platinum® SYBR® Green qPCR SuperMix UDG with ROX reagent (Invitrogen) for quantification of amplicons. The standard PCR conditions were as follows: 50°C (2 min), 95°C (10 min); 40 cycles of 94°C (30 s), 58°C (30 s), and 72°C (1 min), followed by the standard denaturation curve, as performed previously by our group [16 (link)]. The sequences of the primers were designed using the Primer Express software (Applied Biosystems) assuming the nucleotide sequences available in the GenBank database (Table 1). In each reaction the Platinum® SYBR® Green qPCR SuperMix UDG with ROX reagent (Invitrogen), 1 µg/µL of each specific primer, and cDNA diluted 20 times were used. In this study, all data were normalized to beta-actin mRNA. Relative increases in CK and CKR were plotted in comparison to the noninfected control group using 2ΔΔCT method.

To determine tissue parasite load in the infected group, quantitative PCR (qPCR) for parasite quantification was performed as seen previously [5 (link)]. At the end of experimental protocols, a hind paw plantar tissue section was collected, and DNA was extracted using TRIzol reagent following manufacturer’s instructions (Invitrogen, ThermoFisher Carlsbad, CA, USA). The DNA purity was assessed by a spectrophotometer and the ratio (260/280 nm) was between 1.6 and 1.8 for all samples. qPCR was performed using Platinum SYBR Green qPCR SuperMix UDG with ROX reagent (Invitrogen, ThermoFisher) with 100 ng total genomic DNA (gDNA). Parasite quantification was performed with Leishmania specific primers JW11 (forward, 50 -CCTATTTTACACCAACCCCCAGT-30) and JW12 (reverse, 50 -GGGTAGGGGCGTTCTGCGAAA-30). The results were presented as parasite DNA expression, using b-actin as a reference gene to normalize data.

Total RNA from cardiac tissue was isolated using TRIZOL (Invitrogen) and SV Total RNA Isolation System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. cDNA was synthesized using 500 ng of RNA through a reverse transcription reaction (ImProm-IITM Reverse Transcriptase, Promega). Real-time PCR quantitative mRNA analyses were performed in a StepOnePlus Real-Time PCR System (Applied Biosystems) using SYBR Green reagents (Invitrogen) for quantification of the amplicons. The standard PCR conditions were as follows: 50°C (2 min), 95°C (10 min); 40 cycles of 94°C (30 s), 58°C (30 s), and 72°C (1 min); followed by a standard denaturation curve. Primers were designed by using the Primer Express software package v2.0 (Applied Biosystems), based on the nucleotide reference sequences available at GenBank database. Platinum SYBR Green qPCR SuperMix UDG with ROX reagent (Invitrogen), 1 mg/mL of each specific primer and a 1:20 dilution of cDNA were used in each reaction. The mean Ct values from duplicate measurements were used to calculate the expression of the target gene, with normalization to internal controls (GAPDH, β-actin, and HPRT).