RNA Extraction and qRT-PCR Analysis of Leishmania-Infected Mouse Spleens

RNA from the spleens from L. donovani-infected mice was extracted using TRIzol (Thermo Fisher Scientific) and cDNA was synthesized by reverse transcription. The spleens were homogenized with 1 ml TRIzol and φ1.0 stainless steel beads in the 2 ml tube using Micro Smash MS100R (Tomy Seiko) at 4°C and RNA was purified according to the manufacture’s protocol. The concentration of total RNA was measured by DU 730 Life Science UV/vis spectrophotometer (Beckman Coulter). A mixture of 1.25 μM oligo (dT)₁₆, 0.5mM dNTPs (Thermo Fisher Scientific) and 1 μg of total RNA in a tube was incubated for 5 min at 65°C. After adding 5× first strand buffer and 10 mM DTT (Thermo Fisher Scientific), 200 U M-MLV (Thermo Fisher Scientific) was added and the tube was incubated for 50 min at 37°C and 15 min at 70°C. Real-time PCR assay was carried out using 2 μl of cDNA as the template, 10 μl of SYBR Select Master Mix (Thermo Fisher Scientific) and primers listed in S1 Table [11 (link),37 (link)] on the ABI Prism 7000 Sequence Detection System (Thermo Fisher Scientific). Data was analyzed by 2^−ΔΔCt methods and normalized by GAPDH. The thermal cycling conditions for the PCR were 94°C for 10 min, followed by 40 cycles of 94°C for 15 sec and 60°C for 1 min.