Qiamp dna blood kit

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.

DNA for molecular analysis was extracted from 500 μl of each urine, collected without preservatives, using the DNeasy® Blood & Tissue Kit (QIAGEN, S.p.A, Italy) according to the manufacturer's instructions. Blood samples, collected in 4-mL Vacutainer® tubes containing EDTA (BD Becton Dickinson S.p.A, Italia), were centrifuged at 1.376 g/s for 10 min and DNA was extracted from 200 μL of plasma using the DNeasy® Blood & Tissue Kit (QIAGEN, S.p.A, Milan, Italy). PBMCs were isolated from whole blood using the standard Ficoll Hypaque density gradient centrifugation technique (Bøyum et al., 1991 (link)), and the number of viable leukocytes was determined by trypan blue exclusion. PBMCs DNA extraction was performed on 10⁶ cells by the QIAmp® DNA Blood Kit (QIAGEN S.p.A, Milan, Italy) according to the manufacturer's instruction. DNA yield of all biological specimens was determined by measuring its concentration in the eluate by absorbance at 260 nm and then stored at −20°C until use.
Standard laboratory procedures for sterile DNA extraction and PCR were practiced for all specimens.

Genomic DNA was isolated from peripheral blood using the QIAmp DNA Blood kit (Qiagen, USA). The concentration and purity of DNA were determined by measuring absorbance at 260 nm and 280 nm. SNP genotyping was carried out for IRGM gene (rs13361189, rs10065172, and rs4958847), ATG16L1 gene (rs2241880), and TNFRSF1A gene (rs4149570) by real-time PCR using SYBR green-based chemistry. Allele-specific primers were designed and synthesized by introducing mismatches at the 3’ terminal position of all forward primers (S2 Table). Briefly, SNP genotyping reactions were carried out in 20 μl volume containing 10 μl of Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific), 1 μl (20 pmol) each forward and reverse primers and 3 μL of nuclease-free water. Allele-specific RT-PCR thermal cycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of denaturation (95°C, 30 sec), annealing for 30 sec at respective annealing temperature for each primer set and extension (72°C for 30 sec), followed by a melting curve analysis at 65°C to 95°C.

DNA extraction was done using QIAmp DNA blood kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The purity of the DNA was determined by calculating the ratio of absorbance at 260/280 nm. All the purified samples were stored at -80°C until further analyses. The polymerase chain reaction (PCR) was used to detect the selected genetic polymorphisms.

Blood samples (10 ml) were taken by venipuncture from all the study patients on introduction into the study and before the administration of any medication. The blood samples were collected into EDTA Vacutainer® tubes and coded before processing to ensure blinding with respect to sample provenance. The samples were transported at room temperature to the laboratory, centrifuged at 2000 g for 10 min at room temperature, the plasma obtained was distributed in 1 ml aliquots into 1 ml criotubes, and stored at -80°C until needed for processing.
- DNA isolation: DNA from plasma samples (2 ml per column) was obtained using QIAmp DNA Blood kit (QIAGEN Inc., CA) according to manufacturer’s recommendations. A final elution volume was 200 μl and the extracted DNA was quantified spectrophotometrically. The amount of DNA recovered, measured as μg/sample, was 0.431 ± 0.019 (mean value ± standard error of the mean). The fcDNA samples were stored at −80°C until needed for analysis.