Amv rt kit

Total RNA was isolated from Leishmania cells using TRIzol reagent (Life Technologies) and dissolved in nuclease-free molecular grade water in the presence of RNase A Inhibitor (NEB) and stored at −80 °C. Purity of RNA was checked by 260/280 ratio in a spectrophotometer. Followed by DNase I (Roche) treatment, 1 μg of total RNA was denatured and mixed with 1 mM dNTPs, 1× RT buffer and reverse transcriptase, gene-specific primers as recommended in AMV RT kit (NEB), and incubated at 42 °C for 30 min for cDNA synthesis. cDNA preparation confirmed by PCR. Next for quantitative PCR analysis, 100 ng of cDNA and gene-specific primers were mixed with 1× premixed solution Luna qPCR master mix (NEB) of Taq polymerase, dNTPs, and buffer in a 20 μl reaction volume. The RT-PCR analysis was done in a BioRad RT-PCR machine. SYBR green dye binds to double-stranded DNA. The C_t values were calculated as fold change in expression.

RNA was extracted using TRIzol reagent (Invitrogen), and quantified with Nanodrop (Thermo). cDNA was generated by AMV-RT kit (NEB), and a random hexamer primer. Real-time qPCR was carried out on the StepOne Plus system from Applied Biosystems using SYBR mix (Thermo). Primer sequences are listed in Supplementary Table 1.