Plasma was collected before any other handling of the sample. Blood samples were centrifuged at 1200× g for 10 minutes and 2.5 mL of plasma was robotically dispensed into 5 mL tubes using a Xiril robotic workstation (Xiril, Hombrechtikon, Switzerland). The remains of the blood sample were used for antibody screening and maternal serology. The plasma fraction was centrifuged at 2400× g for 20 minutes and subsequently dispensed into two 96 well plates with the Xiril robot, 1 mL of plasma in each plate. One plate was stored at –20°C as a backup; the other was presented to the MagNa Pure 96 Instrument (Roche Holding, Basel, Switzerland) for automated DNA extraction (Viral NA Large Volume Kit; Roche), with a final elution volume of 50 μL.
Viral na large volume kit
Manufactured by Roche
Sourced in Switzerland, Germany
The Viral NA Large Volume Kit is a laboratory equipment product designed for the extraction and purification of nucleic acids (DNA and RNA) from large volume samples, such as those used in viral detection and quantification. The kit provides the necessary reagents and protocols to efficiently isolate high-quality nucleic acids from various biological sample types. The core function of the product is to facilitate the extraction and purification process, enabling users to obtain reliable and consistent results for their downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Magna pure 96 dna, by Roche (5 mentions)
Magna pure 96 system, by Roche (4 mentions)
Magna pure 96 instrument, by Roche (2 mentions)
Magna pure 96, by Roche (1 mentions)
Avl buffer, by Qiagen (1 mentions)
Precellys 24 system, by Bertin Technologies (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Carrier rna, by Merck Group (1 mentions)
Bead mill 24 homogenizer, by Thermo Fisher Scientific (1 mentions)
Magnapure 96 extraction robot, by Roche (1 mentions)
Rna process control kit, by Roche (1 mentions)
9 protocols using viral na large volume kit
1
Automated Blood Plasma Extraction
2
Sanger Sequencing of ALPL, GGPS1, CYP1A1
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Sanger sequencing for the ALPL, GGPS1, and CYP1A1 genes was performed in all patients. Genomic DNA was extracted from peripheral blood using the MagNA Pure 96 DNA and Viral NA Large Volume Kit on the automated DNA extractor MagNA Pure 96 System (Roche Life Science, Switzerland). Standard PCR procedures were performed for Sanger sequencing of all the exons and intron splicing sites of the ALPL (NM_000478), GGPS1 (NM_001037277), and CYP1A1 (NM_000499) genes. Primer sequences are available upon request.
3
Detection of Anaplasma phagocytophilum DNA
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DNA from the EDTA blood samples were extracted on MagNA Pure 96 with MagNA Pure 96 DNA and viral NA large volume kit (Roche Molecular Systems, Inc. Basel, Switzerland) according to the manufacturer’s recommendations. To detect A. phagocytophilum DNA, a quantitative real-time PCR method was performed according to Henningsson et al. 2015. This method amplifies a 64 bp fragment of the gltA gene of the bacterium [43 (link)]. A positive A. phagocytophilum control and a synthetic plasmid (pAP-GltA cloned in pUC57, GenScript Cooperation, Scotch plains, NJ) were used in the qPCR. Nuclease free water was used as negative control.
4
Stool DNA Isolation Protocol
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For DNA isolation, 150–250 mg of stool sample was diluted in 500 μl of AVL buffer (Qiagen, Darmstadt, Germany) and subsequently homogenized using a soil-grinding SK38 kit on the Precellys 24 system (Bertin Technologies, Saint-Quentin, France). Homogenized samples were further extracted on a Magna Pure 96 system (Roche, Basel, Switzerland) using the DNA and Viral NA Large Volume kit (Roche, Basel, Switzerland) according to the manufacturers' protocol.
5
Genomic DNA Isolation and ALPL Gene Sequencing
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Whole blood samples were processed by the Biomedical Diagnostic Centre of the Hospital Clinic of Barcelona, where DNA isolation and ALPL gene sequencing were performed. Genomic DNA was extracted from peripheral blood lymphocytes using the MagNa Pure 96 DNA and Viral NA Large Volume Kit in the automated DNA extractor MagNa Pure 96 System (Roche Life Science, Switzerland) according to the manufacturer's instructions, and the ALPL gene was amplified by PCR. Subsequently, Sanger sequencing was performed using the PCR product as a basis and the truncated sequence NM_000478.5 as a reference to determine the sequence of the coding regions and exon-intron junctions of the ALPL gene. Next, a study of copy number variants was performed by multiplex ligation probe amplification (MLPA) (MRCHolland). The results were analyzed using the SeqPilot program (JSI Medical Systems).
6
Wastewater RNA Extraction Protocol
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A 300 mL 24-hour composite sample of primary post-grit influent or raw wastewater was mixed by inversion and then a 30 mL aliquot was drawn and centrifuged at 4000 ×g for 20 min at 4 °C. The supernatant was removed using a pipette, leaving approximately 500 μL of supernatant with the pellet. The pellet was resuspended and transferred to a 2 mL tube containing 200 μL 0.5 mm zirconia-silicate beads (Biospec, Bartlesville, OK) and 700 μL Qiagen Buffer RLT (Qiagen, Germantown, MD) containing 1% 2-mercaptoethanol. The mixture was processed in a Bead Mill 24 Homogenizer (Fisher Scientific, Ottawa, ON) at 6 m/s for four 30 second cycles. The pelleted debris was removed by centrifugation at 12,000 ×g for 3 min, and up to 1 mL lysate was transferred to a new 2 mL processing plate containing 2 μL of 10 mg/μL carrier RNA (Sigma-Aldrich, Oakville, ON) in each well. RNA was extracted using the Magna Pure 96 DNA and Viral NA Large Volume Kit (Roche Diagnostics, Laval, QC) using the Plasma External Lysis 4.0 protocol as per manufacturer instructions.
7
Influenza A Virus Detection in Avian Samples
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All samples were analyzed at Linnaeus University, Kalmar, as described previously (Wille et al., 2014 (link)). In short, RNA from cloacal and fecal samples was extracted with a MagNA Pure 96 Extraction robot and the viral NA large volume kit (Roche Diagnostics GmbH, Mannheim, Germany). Influenza A viruses were detected using real-time reverse transcriptase PCR (RRT-PCR) assays targeting the matrix gene of the influenza A virus (Wille et al., 2014 (link)). Samples were considered positive for influenza A viruses if the cycle threshold values were <40.
8
Optimizing Viral Nucleic Acid Extraction for CSF
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For optimisation experiments (appendix p 3 ), WNA (representing DNA + RNA) was extracted from phosphate-buffered saline (PBS) seeded with H99 cells (simulated CSF) using the MagNA Pure 96 Instrument (Roche Diagnostics, Mannheim, Germany) and Viral NA Large Volume Kit (Roche Diagnostics). We used the DNA Blood LV 1000 (Roche Diagnostics) and the Pathogen Universal 1000 (Roche Diagnostics) kits for DNA extraction and compared their cycle quantification (Cq) values. Different pretreatment conditions were tested, including untreated (control), bead beating, adding 50 μL of proteinase K and 10 min incubation at 65°C, and a combination of bead beating and adding proteinase K.
For patient CSF screening, WNA was extracted from frozen CSF pellet obtained by centrifugation of 1 mL of CSF. The Pathogen Universal 1000 was used for extraction with 100 μL of elution. The RNA Process Control Kit (Roche Diagnostics) was used as an internal control in RT-qPCR, and a DNA internal control kit (DICR-CY5, Diagenode, Seraing, Belgium) was used in qPCR runs, with a defined virus quantity directly added in the sample before extraction, as recommended.10 (link)
For patient CSF screening, WNA was extracted from frozen CSF pellet obtained by centrifugation of 1 mL of CSF. The Pathogen Universal 1000 was used for extraction with 100 μL of elution. The RNA Process Control Kit (Roche Diagnostics) was used as an internal control in RT-qPCR, and a DNA internal control kit (DICR-CY5, Diagenode, Seraing, Belgium) was used in qPCR runs, with a defined virus quantity directly added in the sample before extraction, as recommended.10 (link)
9
Automated RNA Extraction from Samples
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Each concentrated sample (1 mL) was subjected to RNA automated extraction using MagNA Pure 96 DNA and Viral NA Large Volume Kit in MagNA Pure 96 system Roche (Roche Diagnostics GmbH – Mannheim, Germany), according to the manufacturer's instructions. The extracted RNA was eluted in a final volume of 50 µL of a 60 mM Tris-HCl buffer. Blank control was included during the RNA extraction to monitor any possible cross contamination during samples processing.
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