Taqman gene expression master mix 2x

RNA was isolated with the TRI reagent (Sigma-Aldrich) method. Reverse transcription for cDNA synthesis was performed using Agilent’s AffinityScript Multiple Temperature cDNA synthesis kit. The cDNA was then used for detection of gene expression by real-time PCR using TaqMan^® Gene Expression assays for detection of genes (18S, Hs99999901_s1; S1PR1, Hs00173499_m1; S1PR3, Hs01019574_m1; S1PR4, Hs02330084_s1; S1PR5, Hs00928195_s1; CXCR1, Hs01921207_s1; Applied Biosystems, Woolston, UK), along with TaqMan^® Gene Expression Master Mix 2X (Applied Biosystems) in an Applied Biosystems qPCR machine, with 18S used as the reference gene and data presented relative to CXCR1 mRNA expression.

RNA and protein were isolated with TRIzol (Thermo). Subsequent processing/isolation of RNA and protein were carried out according to the manufacturer’s instructions. RNA sample purity and concentration were assessed by measuring A260/280 spectrophotometrically and were stored at −80 °C. cDNA was synthesized using high capacity cDNA reverse transcription kit (Applied Biosystems) following manufacturer’s instructions. All qPCR primers were purchased from Applied Biosystems and represented probes targeting RIPK1, TAK1, AKT, M-CSF, GM-CSF, IL-8, IL-6, MCP-1, Mip3α, VCAM-1, ICAM-1, cIAP2, and β-actin. The real-time qPCR reactions were performed using Taqman gene expression assays (FAM dye-labeled MGB probes) and Taqman gene expression master mix (2x) (Applied Biosystems) according to manufacturer’s instructions. The real-time qPCR reactions were performed on a Step one plus real-time PCR system (Applied Biosystems). mRNA expression data were normalized to the levels of β-actin as an internal control. Western blot analysis on the stored cell lysates was performed on protein isolated from the same well as the sample used for qPCR, in order to ensure expected changes to RIPK1/TAK1 occurred.

Real-time PCR was performed as originally described by Vallur et al. [31 (link)]. Briefly, TaqMan primers and probes targeting a conserved region of Leishmania REPL repeats (L42486.1) specific to L. donovani and L. infantum were synthesized by Applied Biosystems [31 (link)] and a 20-μL reaction mix prepared containing a 5-μL template, 10 μL of TaqMan Gene Expression Master Mix (2X), 1 μL of pre-ordered primer-probe mix, and PCR-grade water. Amplification was performed on a Biorad CFX96 iCycler system with the following reaction conditions: 10 minutes at 95°C, followed by 45 cycles of 15 seconds at 95°C and 1 minute at 60°C. To quantify the parasite load of each sample, each run included 1 standard curve with DNA concentrations ranging from 10 000 to 0.1 parasites [21 (link)]. Each run also included 1 reaction with molecular-grade water as a negative control. Each DNA sample was evaluated in triplicate. Samples with a cycle threshold (Ct) >40 were considered negative.