Quantification of Sirpa and Actb Expression

RAW264.7 cells were either stimulated with 100 ng/mL LPS or infected with L. donovani (MOI: 50) for 24 h as described above. RNA was extracted using a TRIzol reagent (Invitrogen). The concentration of total RNA was measured using a DU730 Life Science UV/vis spectrophotometer (Beckman Coulter, Chaska, MN, USA), and 4 µg of total RNA was used as the template for the synthesis of cDNA. A tube containing 500 ng of oligo (dT)16 and 10 nmol of dNTPs (Fisher Scientific, Loughborough, UK) with template RNA was incubated for 5 min at 65 °C. Then, 5× first-strand buffer, 200 nmol of DTT (Thermo, Waltham, MA, USA), and 200 U of M-MLV (Thermo) were added, and the tube was incubated at 37 °C for 50 min. The reaction was stopped by incubation for 15 min at 70 °C. The synthesized cDNA was used for the expression analyses of Sirpa and Actb. The designed primers are listed in Table 1 [15 (link)]. A real-time polymerase chain reaction (PCR) assay was conducted using 1 µL of reverse transcription PCR product as the template and 10 µL of SYBR Select Master Mix (Thermo) with the Applied Biosystems QuantStudio 5 Real-Time PCR System (Thermo). Data were analyzed with 2−ΔΔCt methods through normalization with Actb. The thermal cycling conditions were 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min.