Perfecta fastmix 2

Total RNA from either ~ 30 mg piece of frozen kidney cortex or BMDM was isolated using the RNeasy Plus Mini Kit (Qiagen, Germantown, MD, USA) and cDNA was prepared using the High-Capacity RNA-to-cDNA Reverse Transcription kit (Applied Biosystems, CA, USA). Quantitative polymerase chain reaction (qPCR) was performed in triplicate with the Roche Universal Probe Library (UPL) system using a Lightcycler 480 (Roche, West Sussex, UK). For each gene, a 7-point standard curve (1:10–1:640) was generated using a mix of all cDNA samples to be analyzed to which each sample was normalized. Kidney and BMDM cDNA samples were diluted 1:40 in nuclease free water prior to qPCR. Primers for rat Actb, Il1b, Il6, P2rx4, P2rx7, Tbp and Tnf (Supplementary Table 1) were designed with the UPL Assay Design Centre and used at a final concentration of 250 nM with UPL probes (Supplementary Table 1) and PerfeCTa FastMix II (Quanta bio, Beverly, MA, USA). Four different primer sets were used to detect P2rx7 mRNA abundance. Absolute quantification was used to determine each gene expression level and results were normalized to that of the housekeeping gene (Actb or Tbp).

Quantitative qPCR-based amplification of C. parvum-specific heat shock protein 70 (hsp70) was performed as previously reported [25 (link)], with slightly modifications. Briefly, forward primer (CP_hsp70_fwd 5′-aactttagctccagttgagaaagtactc-3′), reverse primer (CP_hsp70_rvs 5′-catggctctttaccgttaaagaattcc-3′), and TaqMan probes (HSP_70_SNA 5′-aatacgtgtagaaccaccaaccaatacaacatc-3′, labelled at the 5′end with the reporter dye FAM (6-carboxyfluorescein) and at the 3′ end with the quencher dye TAMRA (6-carboxytetramethyl-rhodamine)) were purchased from Biomers (Ulm, Germany). The final qPCR reaction volume was 20 μL, containing 1X Polymerase (PerfeCTa FastMix II^®, Quantabio, Beverly, MA, USA), 5 μL DNA template, 0.8 μL of forward and reverse primer (400 nM), 0.4 μL TaqMan probe (200 nM), and 0.2 μL BSA (10 mg/mL). qPCR reactions were performed in triplicate in a Rotor-Gene Q thermocycler^® (QIAGEN, Hilden, Germany) under the following conditions: 95 °C for 5 min, 45 cycles at 94 °C for 15 s, and 60 °C for 60 s. Negative controls consisting of gDNA from non-infected BSI explants, sporozoites (infection doses), and a plasmid standard of target sample template DNA were included in each reaction.

RNA was isolated according to manufacturer’s instructions using TRIzol reagent (Invitrogen). RNA concentration was assessed by Nanodrop 8000 (Thermo Fisher Scientific). cDNA was synthesized using M-MLV reverse transcriptase (Invitrogen) and real-time PCR was performed with PerfeCTa FastMix II (QuantaBio) and the Applied Biosystems StepOne Plus Real-Time PCR system (Applied Biosystems). Gene expression was analyzed by the ΔΔCT method and normalized to TATA-box binding protein as a reference gene. The primers used are listed in Supplementary Table 1.