Subjects (n = 348) were recruited from National Heart Centre Singapore and via advertisement at the MRC Clinical Sciences Centre, Imperial College London. Samples for WGS (n = 8) were obtained from National Cancer Centre Singapore, National University Hospital Singapore. All participants gave written informed consent, and study protocols were approved by the local institutional ethics committees and carried out in accordance with local Tissue Acts, as appropriate. Genomic DNA was extracted from blood using Prepito DNA Blood 600 kit (Perkin Elmer, MA) (targeted sequencing), EZ1 DSP DNA blood 48 kit (Qiagen, Netherlands) (WES) or QIAsymphony DNA kit (Qiagen, Netherlands) (WGS) following manufacturer’s protocols. Quality and quantity of extracted DNA were assessed by an ultraviolet-visible spectrophotometer.
Qiasymphony dna kit
Manufactured by Qiagen
Sourced in Netherlands, United States, Italy
The QIAsymphony DNA kit is a laboratory equipment product designed for the automated extraction and purification of DNA samples. It offers a standardized and efficient method for isolating DNA from a variety of sample types, ensuring consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Massarray system, by Agena (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Qiasymphony workstation, by Qiagen (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Prepito dna blood600 kit, by PerkinElmer (1 mentions)
Qiamp dna mini kit, by Qiagen (1 mentions)
Ficoll paque plus gradient, by GE Healthcare (1 mentions)
Qiasymphony rna kit, by Qiagen (1 mentions)
Sepmate tube, by STEMCELL (1 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (1 mentions)
Qiamp dna tissue kit, by Qiagen (1 mentions)
8 protocols using qiasymphony dna kit
1
Large-Scale Genomic DNA Extraction
2
EGFR Mutation Detection by MALDI-TOF MS
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The EGFR mutations in tissue specimens were detected by matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI–TOF MS) as described previously [58 (link),59 (link)]. Briefly, DNAs were extracted from the paraffin-embedded tissues either by the QIAmp DNA Mini kit or the automated QIAsymphony extraction system with the QIAsymphony DNA kit (Qiagen, Valencia, CA), following the manufacturer’s protocol. MALDI–TOF MS was used to detect the genetic alterations of EGFR. The analysis of the results was performed according to the manufacturer’s protocol for the MassARRAY system (Agena Bioscience, San Diego, CA, USA).
3
Genetic Profiling of Cattle Breeds
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A total of 93 living Rouge-des-Prés cattle were tested for SLC25A46 and MAN2A1 polymorphisms, including 27 with clinical symptoms of distal axonopathy (with or without subsequent histopathological confirmation). In addition, 31 more historical Rouge-des-Prés animals were also tested, as well as 321 other cattle from 12 French breeds. For all of these, DNA was extracted from blood samples (Genisol Maxi-Prep kit or QIAsymphony DNA Kit (Qiagen)).
4
Isolation and Culture of PBMCs and LCLs
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Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples of individuals P1 to P22 and their biological parents using a Ficoll-Paque Plus gradient (GE Healthcare Life Sciences) in SepMate tubes (STEMCELL Technologies) according to the manufacturer’s protocols. LCL derived from individuals P23 to P39 were expanded in RPMI 1640 medium supplemented with GlutaMAX (Thermo Fisher Scientific), 10% fetal bovine serum, 1% penicillin, and 1% streptomycin at 37 °C. LCL cultures of each individual were split into three flasks and cultured separately for at least 1 week to obtain technical replicate samples for RNA isolation. Genomic DNA was isolated from the PBMCs or LCL using the QIASymphony DNA kit (Qiagen). Total RNA was isolated using the QIAsymphony RNA Kit (Qiagen), and RNA quality (RIN > 8) was determined using the Agilent RNA 6000 Nano Kit.
5
EGFR Mutation Detection in Paraffin-Embedded Tumor Tissues
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Genomic DNA from the paraffin-embedded tumor tissues was extracted using either the automated QIAsymphony extraction system with a QIAsymphony DNA kit (Qiagen, Valencia, CA, USA) or QIAmp DNA Tissue kit (Qiagen). To further classify the DNA genome into MT or WT EGFR, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) was used as described previously [24 (link),25 (link)], and the results were analyzed using the MassARRAY system (Agena Bioscience, San Diego, CA, USA).
6
DNA Extraction from Blood Samples
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The genomic DNA of each patient was extracted from peripheral blood by QIAsymphony workstation and QIAsymphony DNA kits (Qiagen, Milan, Italy). For all DNA samples collected, the concentration was measured with Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The standard DNA samples were stored in refrigeration at −20 °C.
7
Whole-Exome Sequencing Variant Analysis
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Buccal swabs were obtained from all patients (n = 30) for whole-exome sequencing (WES). Samples were sent for automated genomic DNA extraction (QIAsymphony DNA kits, QIAGEN). Target regions were captured using a custom library preparation kit (Illumina). Variant calling was then performed and retrieved variants analyzed using Varstation.6 Variants were classified as per the American College of Medical Genetics and Genomics.6
8
Whole Exome Sequencing from Blood
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DNA from peripheral blood was extracted using the QIAsymphony workstation and the QIAsymphony DNA kits (Qiagen). DNA quantity and quality were assessed with Qubit (Thermo Fisher Scientific) and agarose gel. For each patient, 1 ug of genomic DNA was sent to Macrogen Inc company (Korea, Seoul) for whole exome sequencing using Illumina Platform. The Human All Exon V6 Library kit (Agilent) was employed to select the target regions (60.5 Mb), whereas the NovaSeq 150pb PE sequencing kit was chosen for sequencing at an average coverage of 100Â.
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