Quantstudio design and analysis software v1

All the strains of L. monocytogenes tested in the current study were serogrouped following the method described by Vitullo et al. (2013 (link)). Briefly, the experiments consisted of multiplex qPCR reactions that were performed in a final reaction volume of 25 μL with the following components: 12.5 μL of NZYSupreme qPCR Probe Master Mix 2X (NZYTech, Lisbon, Portugal) 300 nM primers and 100 nM probes for lmo0737, ORF2819, and ORF2110, while for lmo1118, 1,000 nM primers and 200 nM probe were added. A total of 5 μL of template DNA was loaded, and the remaining volume was filled with sterile MilliQ water. The thermal profile selected included a hot-start step at 95°C for 5 min, followed by 40 cycles of dissociation at 94°C for 5 s, and annealing-extension at 60°C for 30 s. These experiments were performed in a QuantStudio 5 Real-Time PCR System, with the QuantStudio™ Design and Analysis Software v1.4.3 (Applied Biosystems™). The sequences of all the primers and probes are provided in Table 2.

The multiplex qPCR assay developed by Roumani et al. was introduced for sample pre-screening prior to MinION sequencing (Roumani et al., 2021 (link)). This assay targets the hly gene of L. monocytogenes and also includes a competitive internal amplification control, see Table 2 for detailed sequences. The reaction mixture consisted of 200 nM of forward and reverse primers, 150 nM of the probe, 100 nM of IAC probe, 1,000 copies of IAC DNA, 10 μL of TaqMan^®Fast Advanced Master Mix (Applied Biosystems™, Foster City, CA, USA), 3 μL of template, and the remaining volume up to 20 μL was filled with sterile MilliQ water. The “Fast” run mode was selected, and the thermal profile consisted of an initial UDG treatment at 50°C for 2 min, hot-start activation at 95°C for 2 min, 40 cycles of dissociation at 95°C for 1 s, and annealing-extension at 63° C for 20 s. All the qPCR experiments were performed in a Quant Studio 5 Real-Time PCR System, with the QuantStudio™ Design and Analysis Software v1.4.3 (Applied Biosystems™).

RNA extraction was performed using the MicroElute Total RNA kit (Omega Biotek, Norcross, GA, USA, R6831-02) according to the manufacturer’s recommendations. Purified RNA was used to synthesize the first strand cDNA using Superscript II (Invitrogen) and random hexamers. RT-qPCR analyses were performed with 4 ng of cDNA as template, using a SYBR Green Mix (Applied Biosystems, Foster City, CA, USA, 100029284) and a QuantStudio 3 (Applied Biosystems) real-time PCR System.
Relative quantitative RNA was normalized using the housekeeping gene β-actin. Primers were designed using NCBI Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Accessed date: 1 December 2020). Analysis of the results was performed using QuantStudio Design and Analysis Software v1.4 (Applied Biosystems) and relative quantification was performed using the DDCt method using β-actin as reference.

Brain tissue (whole brain) was placed into 1 mL of RNAlater reagent (Qiagen, Germantown, MD, USA) and stored at 4 °C. Total RNA was extracted using the RNeasy kit (Qiagen, Germantown, MD, USA) and measured using standard spectrophotometric parameters on NanoDrop (Thermo-Fisher, Waltham, MA, USA). RNA (1 μg) from each sample was retrotranscribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). cDNA samples (2 μL) were used as the template for amplification reactions performed with the TaqMan Fast Advanced Master Mix and Vegfa or Epo TaqMan assay FAM and Hprt TaqMan assay VIC in a QuantStudio 3 Real-Time PCR System (all from Applied Biosystems, Waltham, MA, USA). Quantitative PCR data analysis was performed with QuantStudio Design and Analysis software v1.4.3 (Applied Biosystems, Waltham, MA, USA).

Array-based semi-quantitative real-time RT-PCR was used to identify the idoneous housekeeping genes for the tissues analyzed.
For cDNA synthesis, 1 μg of RNA was reverse transcribed by random priming with SuperScript Vilo MasterMix (Invitrogen, USA) following manufacturer’s instructions, and then cDNA samples were tested by the Taqman Array Human Endogenous Control (ThermoFisher Scientific, Waltham, MA, USA) containing 32 housekeeping candidates. About 10 ng of cDNA per sample was amplified in triplicate in a 20 μL reaction volume in presence of Taqman Universal Master Mix II, no UNG (ThermoFisher Scientific, USA). Reaction conditions: 10′ at 95 °C followed by 40 cycles (15″ at 95 °C, 60″ at 60 °C). Results were visualized by QuantStudio Design and Analysis Software v. 1.5.2 (ThermoFisher Scientific, USA) and by the RT2 Profiler PCR Array data analysis tool (Qiagen, Hilden, Germany). Genes with the higher consistency across tissues were evaluated by comparing cycle threshold (Ct). Relative expression was obtained by normalization to one of the 32 putative housekeeping analyzed.
To evaluate results, mean relative expression from the 3 AIS cases was used.

Ordinary reverse transcription real-time PCR (qRT-PCR) assays were performed in 20 μl of total reaction volume. Each reaction tube comprised 5 μl of RNA solution, 10 μl of Luna universal probe one-step RT-qPCR mixture (New England Biolabs, Beverly, Massachusetts, USA), 1 μl of Luna warm start RT enzyme Mix (New England Biolabs), 400 nmol/l of primers HOT_Spike_Fw/HOT_Spike_Rv, and 200 nmol/l of probe P_LANL_4.1 (Table 1). The same procedure was used for the qRT-PCR assay oriented to N gene, but the oligonucleotides were N3-F/N3-R (primers) and N3-P (probe; Table 1). The real-time PCR cycler used was Applied Biosystems QuantStudio 1 (Thermo Fisher, Waltham, Massachusetts, USA), and the temperature program consisted of the following steps: 55 °C for 10 min, 95 °C for 1 min, 45 cycles of 95 °C for 10 s, and 60 °C for 1 min. The fluorescence signal in FAM channel was collected at the end of each cycle. Each run contained positive (ESRM or ENRM) and negative controls. Data were collected and analyzed using QuantStudio Design and Analysis Software v1.5.2 (Thermo Fisher). Cycle threshold (C_t) values were calculated at the automatic threshold setting. The fluorescence signal in positive samples showed a typical S-shaped amplification curve, and a sample was considered positive when C_t ≤ 38.