Magna pure lc system

RNA isolation and RT-PCR were performed as described previously (14 (link), 15 (link)). Briefly, RNA from swabs and tissue suspensions was isolated by using a MagNaPure LC system with the MagNaPure LC total nucleic acid isolation kit (Roche Diagnostics, Almere, the Netherlands). Real-time RT-PCR assays were performed on an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) by using the TaqMan EZ RT-PCR core reagents kit (Applied Biosystems, Nieuwerkerk a/d IJssel, the Netherlands) according to the manufacturer’s instructions. For each run, the samples were prepared and processed in parallel with several negative and positive control samples. Virus titers were determined by serial 10-fold dilution of the homogenized tissue samples and swabs on MDCK cells, as described elsewhere (14 (link), 15 (link)). Virus titrations were performed in triplicate.

DNA was extracted from EDTA-anticoagulated blood samples using the MagNA Pure LC system (Roche Diagnostics, Basel, Switzerland) following the manufacturer’s instructions.
Using online search engines such as PubMed, Ensemble, and HuGE navigator, a systematic literature search was conducted to identify SNPs statistically significantly associated with LTPA. The search time frame related to the present study was until 5 August 2019. Keywords and their combinations used in the search: leisure time physical activity, recreational physical activity, genetics, genome-wide association study (GWAS), candidate gene, genotype. In the selection of SNPs, particular attention was focused on the results of the three GWAS [14 (link),15 (link),73 (link)] and a candidate gene study [82 (link)], which were the most relevant in this field.
The literature search identified a total of ten SNPs, and these were genotyped using the MassARRAY platform (Sequenom Inc., San Diego, CA, USA) with iPLEX Gold chemistry in the Mutation Analysis Core Facility (MAF) of the Karolinska University Hospital, Sweden. The MAF conducted validation, concordance analysis, and quality control according to their protocols. The Hardy-Weinberg Equilibrium (HWE) and linkage disequilibrium (LD) structure of the genotyped SNPs were calculated by Haploview software (version 4.2; Broad Institute; Cambridge, MA, USA).

A total of 96 BC index patients with a high risk of BC were selected retrospectively. We extracted DNA from peripheral blood lymphocytes sample for their BRCA1/2 study and the excess was stored in the Health Care Biobank. The study was performed in genomic DNA extracted from 500 μL of whole blood using the MagNA Pure LC DNA Isolation Kit, large volume (Roche, Mannheim, Germany) automated in the MagNA Pure LC System (Roche), according to the manufacturer’s protocol.

Nucleic acids were extracted from all clinical samples using the MagNa Pure LC system (Roche Life Science, North Ryde, NSW, Australia), according to the manufacturer’s instructions. Previously described PCR/RT-PCR (real-time and conventional) methods were used to screen for a large range of viral and bacterial pathogens (Supplementary Table S1), including the viruses: JEV, DENV1-4, chikungunya virus (CHIKV), flavivirus universal, EV-A71, enterovirus universal, influenza A viruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex virus (HSV), varicella-zoster virus (VZV); and the bacteria: S. pneumoniae, H. influenzae, N. meningitidis, Orientia tsutsugamushi and Streptococcus suis. Mycobacterium tuberculosis testing was also undertaken using GeneXpert for the first 40 patients enroled with a high clinical suspicion of tuberculosis AME, but testing was discontinued due to the requirement for high volumes of CSF and the absence of positive results. Testing of other pathogens (for example, rabies, measles and mumps) was conducted if the clinical presentation was suggestive of a particular infection.

Total nucleic acid was isolated from serum samples using the robotic Roche MagNA Pure LC system (software version 3.0.11) and the MagNA Pure LC Total Nucleic acid isolation kit (Roche Diagnostics GmbH, Mannheim, Germany), and eluted in 50 μl of lysis buffer according to the manufacturer’s instructions. Nearly full-length genomes of HBV genotype B (GT/B) and genotype C (GT/C) strains were amplified using two rounds of PCR as previously described^{47 (link)}. Further details can be found in “SI Methods”.

DNA was isolated using a MagNA Pure LC system (Roche Diagnostics, Basel, Switzerland) with a MagNA Pure LC DNA Isolation Kit—Large Volume according to the manufacturer’s instructions. Extracted DNA was eluted in 200 µL MagNA Pure LC DNA Isolation Kit—Large Volume elution buffer.
A systematic literature review on the PubMed, HuGE Navigator and Ensembl databases was conducted to identify single nucleotide polymorphisms (SNPs) in CETP and LIPC genes, which are most strongly associated with cholesterol metabolism. The literature search resulted in the selection of 5 SNPs in CETP and 6 SNPs in the LIPC gene. The genotyping was conducted by the Mutation Analysis Core Facility at the Karolinska University Hospital, Sweden.
Genotyping was performed on a MassARRAY platform (Sequenom Inc., San Diego, CA, USA) with iPLEX Gold chemistry. Validation, concordance analysis and quality control were conducted by the facility according to their protocols. Successful genotyping was obtained in 2518 DNA samples (746 Roma and 1772 Hungarian general samples).
More details on the study design, sample populations, selection process of SNPs and genotyping are described in our previous research paper [22 (link)].

Venous blood was taken with parents agreement after clinical examination of the children in the study center. The heparin blood for the determination of cytokines and neuropeptides was processed after a maximum of 4 hours storage at room temperature; the blood samples were stored at –80 °C until further analysis was carried out. The neuropeptides VIP, SOM and SP were determined using ELISA (Phoenix Europe, Karlsruhe, Germany) and the cytokines (IFN-γ, IL-4, IL-5 and IL-9) using cytometric bead array (CBA, BD Bioscience, Heidelberg, Germany) according to the manufacturers instructions and as described by Herberth and colleagues [14 (link)].
The transcription factors (GATA3, Tbet, FOXP3) and the regulatory factors (SOCS1, SOCS3) were determined after RNA extraction from 1 ml of heparinized blood (MagNA Pure LC System and MagNA Pure LC mRNA Isolation Kit, Roche Applied Science, Mannheim, Germany) using real-time PCR (Lightcycler, LC search Primer Kits, Roche Applied Science, Mannheim, Germany) as described by Herberth and colleagues [14 (link)].