Ez1 advanced xl

Nucleic acid was extracted from samples on an EZ1® Advanced XL workstation using the EZ1 Virus Mini Kit v2.0 (Qiagen, Hilden, Germany). From 2009 to 2015, HEV was detected by RT-qPCR according to Wenzel et al. [24 (link)]. In 2016, the RT-qPCR was replaced by a protocol by Jothikumar et al. with a modified probe [25 (link),26 (link)]. Both assays were calibrated against the WHO International Standard (code number 6329/10) and HEV RNA was quantified as International Units per mL (IU/mL).

Genomic DNA was isolated from peripheral blood cells using an automated DNA extraction system (EZ1 Advanced XL, Qiagen, Hilden, Germany). DNA concentration and purity were evaluated by absorbance methodology using a NanoDrop 1000 Spectrophotometer V3.7 (Thermo Fisher Scientific, Waltham, MA, USA). The following allelic variants were investigated: NAT2*5 c.341 T > C (rs1801280), NAT2*6 c.590 G > A (rs1799930), NAT2*7 c.857 G > A (rs1799931), and NAT2*14 c.191 G > A (rs1801279). All genotypes were determined by real-time PCR using the LightSNiP (TIB-MolBiol, Berlin, Germany) on a LightCycler 480 (Roche, Basel, Switzerland). Genotyping performance was estimated through the use, in each analysis, of known-genotype internal quality controls.
Based on the presence of the NAT2 allelic variants, the patients were grouped into three acetylator phenotypes (fast, intermediate, and slow) following the PharmGKB classification (available at https://www.pharmgkb.org/vip/PA166170337, accessed on 15 April 2023).
All patients provided additional written signed consent for the pharmacogenetic analyses.

Metagenomic sequencing was performed directly from the blood culture fluid as previously described [6 (link)]. Briefly, 200 µL of blood culture material was obtained and DNA was extracted using Qiagen EZ1 Blood and Tissue Kit on the EZ1 Advanced XL instrument per the manufacturer’s recommendations (Qiagen, Valencia, CA, USA). Mycobacterial specimens were first heat-inactivated (100 °C for 30 min) and an additional bead-beating step was performed for the mechanical disruption of the cell wall. Bead-beating step was also performed for fungal specimens. Libraries were prepared and sequenced on the Illumina MiSeq System (Illumina, San Diego, CA, USA) using a 2 × 250 bp standard protocol per the manufacturer’s recommendations. Detailed steps regarding library preparation and sequencing have previously been described in our study on the validation and implantation of whole genome sequence-based bacterial identification in the clinical microbiology laboratory [8 (link)].

For the experiments, 100 pM LH and hCG dose maximally activating cell signaling [9 (link),14 (link),16 (link),28 (link)] was used for gene expression analysis. Cells were seeded in 24-well plates (1 × 10⁵ cells/well) and maintained under continuous stimulation by LH and hCG. Gonadotropins are highly stable in culture media and are measurable after several hours, as previously observed [14 (link)]. Samples were lysed and subjected to RNA extraction using the automated extractor EZ1 Advanced XL (Qiagen, Hilden, Germany). Equal amounts of total RNA were retro-transcribed by iScript reverse transcriptase (Bio-Rad Laboratories Inc.) according to the following protocol, as previously described [18 (link)]: 25 °C for 5 min; 42 °C for 30 min; 85 °C for 5 min. Quantitative real-time PCR was performed in triplicates using specific gene primer sequences (Table 1) and settings: 95.0 °C for 30 s; 95.0 °C for 3 s and 45 cycles; 57.0 °C for 30 s. Normalized gene over the ribosomal protein S7 (RPS7) gene expression was evaluated using the 2^-ΔΔCt method [52 (link)]. Real-time PCR method and primer sequences were validated previously [14 (link),16 (link),28 (link)].