Qiasymphony dsp dna mini kit 192

DNA was extracted from whole blood using the EZ1 DNA Blood 350 µl Kit or QIAsymphony DSP DNA Mini Kit (192) (Qiagen, Hilden, Germany). Whole gene characterization (5′UTR to 3′UTR) for HLA-A, B, C, DQA1, DPA1, and DQB1 and key region characterization (exon 2 to 3′UTR) of HLA-DRB1, DRB3, DRB4, DRB5, and DPB1 was performed using the Holotype HLA kit version 2 (Omixon, Budapest, Hungary). Libraries were sequenced using MiSeq Sequencer (Illumina, California, USA) and sequence data were analyzed using the HLA Twin version 2.5 (Omixon, Budapest, Hungary).
HLA eplets were obtained from allelic data using the computer algorithm HLAMatchmaker v02 for HLA class I and v02.2 for HLA class II genes (www.epitopes.net) which uses a database of alleles to define a string of eplets for each allele. Only eplets registered to be experimentally confirmed by antibody testing (antibody-verified eplets) were used in this analysis.
Allele or eplet carrier rates (relative frequencies) were calculated respectively as the proportion of subjects expressing a specific allele or eplet within the total study sample. Composite genotypes (HLA types) and their corresponding epitypes were calculated respectively as the numbers of subjects within the sample expressing each unique combination of alleles or eplets at all the 11 allelic HLA gene loci.

Genetic testing was performed for RNF213 (Ring finger protein 213) gene founder variant p.R4810K in all identified patients from our institution.
The deoxyribonucleic acid (DNA) isolation was performed from ethylenediamine tetraacetic acid (EDTA) blood using QIAsymphony DSP DNA Mini Kit (192) (Qiagen), in combination with the QIAsymphony SP instrument (Qiagen). Polymerase chain reaction (PCR) amplification was performed employing the HotStarTaq DNA Polymerase kit (Qiagen). Amplified PCR products were cleaned up using the illustra™ ExoProStar™ 1-STEP kit (VWR). The cleaned-up PCR products were cycle sequenced employing the BrilliantDye™ Terminator v1.1 Cycle Sequencing Kit (NimaGen), followed by a bead-based clean-up of the cycle PCR products and subsequent capillary electrophoresis on the 3730xl DNA Analyzer. Generated.ab1 files were analyzed with the aid of the SeqPilot software. The used oligonucleotides (primers) had the following base sequence:
131087_RNF213-E60F: ACGCTGCATCACAGGAAA
131088_NF213-E60R: GCTTAGGCTATAGAGCACCCA.

Whole blood was extracted using the EZ1 DNA Blood 350 µL Kit (Catalog 951054) or QIAsymphony DSP DNA Mini Kit (192) (Catalog 937255) (Qiagen, Germany). Both methods use magnetic beads to isolate DNA from leukocytes where the resulting DNA is eluted in water or a buffer. DNA was quantified using the Qubit Fluorometer (ThermoFisher, United States) and diluted to 10–35 ng/μL for sequencing.