Qiamp dna blood mini kit

CirDNA from plasma, serum or cell medium was extracted using the QIAmp DNA Mini Blood kit (Qiagen), used according to the “Blood and body fluid protocol,” using 80 µl of elution volume. DNA extracts were kept at − 20 °C until used.

All blood samples were collected in 4-milliliter EDTA tubes. The blood was then centrifuged at 1200g at 4°C for 10 minutes. The supernatants were isolated in sterile 1.55 mL Eppendorf tubes and centrifuged at 16,000g at 4°C for 10 minutes. Afterward, the plasma was either immediately used for DNA extraction or stored at –20°C. CfDNA was extracted from 1 mL of plasma using the QIAmp DNA Mini Blood kit (Qiagen) according to the “Blood and body fluid protocol.” DNA extracts were kept at –20°C until used. The preanalytical conditions we followed are described (45 (link)).

The 20 µl of chromatin equivalent to 200 ng of genomic DNA was added to 5 aliquots of 500 µL of blood EDTA, plasma EDTA, or serum. These samples were incubated at 37 °C for 0 min, 30 min, 2 h, 8 h, and 24 h. After incubation, the plasma was isolated from blood EDTA as described above. Then, cfDNA was extracted from 0.5 ml of plasma or serum using the QIAmp DNA Mini Blood kit (Qiagen, CA), according to the “Blood and body fluid protocol” and our own detailed protocol [38 (link)]. CfDNA samples were kept at 20 °C until use. Due to EDTA-associated calcium chelation inhibitory effect on nucleases, comparison of plasma vs serum incubation may help in estimating nuclease role in degrading gHMW DNA.

Samples of peripheral venous blood (5 ml) were collected from patient and his parents for genomic DNA extraction using a QIAmp DNA Mini Blood Kit (Qiagen, Hilden, Germany). To reveal the disease causing mutation in this family, whole exome sequencing approach was performed on this family. Briefly, the libraries for whole exome sequence were established from the DNA samples using an exon capture kit (SureSelect ver. 6 + UTR, Agilent Technologies), according to the manufacturer’s instructions. The exons were sequenced as 100-basepairs paired-end reads by an Illumina HiSeq2500 (Illumina). The original sequencing data were processed by Illumina Basecalling Software 1.7 and compared with NCBI HUMAN genome DNA reference sequence (NCBI Build 37.1). Single nucleotide variation (SNV) information is analyzed using SOAP software (http://soap.Genomics.org.cn). The information related to Inserts and deletions (Indel) is analysed using BWA software (http://bio-bwa.sourceforge.net/) to obtain all the mutations occurring in the DNA sequences of the sample. The common variants (MAF > 1%) that appear in the database (DB135) are filtered out, and then those that have no effect on the structure and function of the protein are filtered out. Candidate pathogenic gene variants were obtained by filtration.

Our study conformed to the Declaration of Helsinki and was prospectively reviewed and approved by the ethics committee of Ningxia Eye Hospital. Written informed consent was obtained from all participants before their enrollment. Eighteen patients and 18 unaffected family members from family DC were included in the study (Figure 1). Medical records from each participant were reviewed. Routine ophthalmic examination was conducted for all participants, and all patients received comprehensive ophthalmic examinations, including best-corrected visual acuity, slit-lamp examination, visual field test, funduscopic evaluations, and electroretinograms. Fundus autofluorescence, fundus fluorescein angiography, optical coherence tomography, and electroretinography were performed when possible. Another 423 unrelated Chinese controls free of maculopathy and other major ocular diseases were also recruited. Samples of peripheral venous blood (5 ml) were collected from each participant for genomic DNA extraction using a QIAmp DNA Mini Blood Kit (Qiagen, Hilden, Germany).

As part of our study, we also investigated the kinetics of in vitro degradation by serum nucleases of the NET produced in the previous part of the study where NET induction was studied. This allows a direct comparison of the levels of NET formation vs production. To do so, the previously obtained supernatants of PMA-stimulated (5 h) neutrophils from a male individual were incubated with a fresh female serum for various time periods. We also used the X/Y differentiation to distinguish NET-derived male DNA from female cirDNA derived from serum. The qPCR analysis was performed targeting the SRY gene on the Y chromosome, and sWGS results took into consideration only reads aligning on the Y chromosome.
The 200 µl of supernatant of the PMA-stimulated neutrophils (5 h) equivalent to 50 ng of genomic DNA was added to aliquots of 500 µL of plasma or serum. These samples were incubated at 37 °C for 5 min, 10 min, 20 min, 30 min, 2 h, 8 h, and 24 h for NET degradation in serum, with 24 h incubation in plasma used as a control. After incubation, cfDNA was extracted from 0.5 ml of plasma/serum using the QIAmp DNA Mini Blood kit (Qiagen, CA), performed according to the “Blood and body fluid protocol” and our own detailed protocol [38 (link)]. CfDNA samples were kept at − 20 °C until use.