Steponeplus cycler

RNA from cells or pieces of liver was purified by the Rneasy Mini Kit from Qiagen according to manufacturer's instructions. Concentration and purity were measured on the Nanodrop (Eppendorf). cDNA was generated using the Quantitect^® Reverse Transcription Kit (Qiagen) according to manufacturer's instructions. Quantitative real‐time RT–PCR (qRT–PCR) was performed on an ABI StepOnePlus cycler using TaqMan probe sets purchased from Applied Biosystems.

Knockdown of miR-71 and RT-qPCR (retrotranscription followed by real time polymerase chain reaction) was performed. For this, miRNA cDNA synthesis was performed as in Macchiaroli et al. [16 (link)] using 5 ng of input RNA. PCR was performed in a StepOne Plus cycler (Applied Biosystems, U.S.A.). The PCR mix consisted of 5 x HOT FIREPol EvaGreen qPCR Mix Plus 2 μl (1x), 0.4 μl (200nM) of each primer, 5.2 μl of water DNAse free and 2 μl of diluted cDNA. The cycling conditions were: 15 min at 95°C, followed by 40 x (15 s at 95°C, 20 s at 60°C) and a final step of 30 s at 72°C. Data collection was performed at 72°C. For primer design, the mature miRNA sequences identified in this work were used (S2 Table). In order to find a reference gene for normalization of the qPCR data, those miRNAs that showed high expression in primary cells of E. multilocularis were evaluated. The data were analysed by two programs: NormFinder [28 (link)] and BestKeeper [29 (link)]. The miR-4989 was chosen as reference gene since it fulfilled the stability requirements: i) low stability index according to NormFinder analysis ii) Cp standard deviation ≤0,5 with coefficient of correlation ~1 according to BestKeeper analysis (S1 Fig). Primer sequences, amplicon size and PCR efficiency values for all the primers are described in S2 Table.

Initially, isolated neutrophils were treated with PP13 (3 μg/ml) or left untreated for 1 h. Total RNA was isolated from 3 × 10⁶ neutrophils using the RNeasy Mini Kit (Qiagen). TaqMan RT-PCR was performed utilizing the Applied Biosystems StepOne Plus cycler (Applied Biosystems) and TaqMan Gene Expression Assay primer and probe sets (Applied Biosystems) for TNF (HS01113624_g1), SERPINB1 (HS00961948_m1), and GAPDH (HS99999905_m1).

RD cells were seeded at 1 × 10^∧5 cells per well in 24-well plates and subsequently infected with the wild-type (WT) E7 or E7 R2 mutants at a multiplicity of infection (MOI) of 0.01 or 1000 E7 RNA copies per cell. One hour post infection the inoculum was removed and cells were washed with phosphate buffered saline (PBS) before adding 500 μl cell culture medium. At the indicated times post infection the cell culture medium was aspirated and stored at −80°C. Where applicable, cells were lysed in 300 μl RLT lysis buffer and stored at −80°C before RNA isolation with the RNease kit (Qiagen). Viral titres were determined in an end point dilution assay (EPDA) by determining the tissue culture infectious dose 50% (TCID50) in RD cells.
Total RNA was harvested according to the manufacturer’s protocol (RNeasy, Qiagen). In a one-step reaction Quantifast Sybr green (Qiagen) total RNA was reverse transcribed with gene specific primers amplifying either E7 (primer pair E7 5’-UTR) or the internal control GAPDH (Supplementary file 1A) using the Stepone plus cycler (Applied Biosystems).

RNA preparation was carried out in cells by direct lysis using the Trizol–Chloroform method, following the manufacturer’s protocol.
Reverse transcription was carried out using the MMLTV reverse transcriptase kit from Invitrogen (Thermo-Fisher, Bd Sébastien Brant, Parc d’Innovation, France).
The expression of the CD24 gene was quantified by TaqMan RT-PCR utilizing the Applied Biosystem StepOne Plus cycler (Applied Biosystems, Austin, TX, USA) and TaqMan Gene Expression Assay with primer and probe sets (Applied Biosystems) for CD24 (Hs02379687_s1).
HPRT and WYHAZ (ABI, Branchburg, NJ, USA) were used to standardize the expression level. The relative amount of CD24 was calculated by employing the comparative CT method (2^−ΔΔCT) [49 (link)]. Amplification was performed for the JEG-3 and BeWo cells transfected by STOX1-A or STOX1-B polymorphic variants compared with the empty expression vector used as a mock control.