DNA extraction and SNP genotyping was performed according to the manufacturer’s instructions in the laboratories and under the supervision of the Department of Clinical Pharmacology, University Medical Center Goettingen, Germany.
Whole blood samples were drawn from all study subjects within 72 h after sepsis onset. The extraction of genomic DNA was performed using either the QIAmp® DNA Blood Kit in QIAcube®, the EZ1® DNA Blood Kit in BioRobot EZ1® or the AllPrep DNA Mini Kit (all from Qiagen, Hilden, Germany), as previously described [21 (link),24 (link),43 (link)]. Quantity and quality of the extracted DNA were tested by spectrophotometric measurement.
The LAG-3 rs951818 was genotyped in all samples through TaqMan polymerase chain reaction (PCR) using the appropriate predesigned TaqMan® SNP Genotyping Assay C___8921385_10 (Thermo Fisher Scientific, Waltham, MA, USA) and a 7900HT Fast-Real-Time PCR System (Life Technologies, Darmstadt, Germany) as well as 7900HT Fast-Real-Time PCR System software (SDS v2.4.1 for Windows 7, Applied Biosystems, Foster City, CA, USA). Over 20% of the samples were genotyped in duplicate to increase reliability.
Whole blood samples were drawn from all study subjects within 72 h after sepsis onset. The extraction of genomic DNA was performed using either the QIAmp® DNA Blood Kit in QIAcube®, the EZ1® DNA Blood Kit in BioRobot EZ1® or the AllPrep DNA Mini Kit (all from Qiagen, Hilden, Germany), as previously described [21 (link),24 (link),43 (link)]. Quantity and quality of the extracted DNA were tested by spectrophotometric measurement.
The LAG-3 rs951818 was genotyped in all samples through TaqMan polymerase chain reaction (PCR) using the appropriate predesigned TaqMan® SNP Genotyping Assay C___8921385_10 (Thermo Fisher Scientific, Waltham, MA, USA) and a 7900HT Fast-Real-Time PCR System (Life Technologies, Darmstadt, Germany) as well as 7900HT Fast-Real-Time PCR System software (SDS v2.4.1 for Windows 7, Applied Biosystems, Foster City, CA, USA). Over 20% of the samples were genotyped in duplicate to increase reliability.
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