Puregene blood core kit

In total, 166 samples from patients diagnosed with diabetic and idiopathic neuropathy were tested by MIPs-NGS and TSCA-NGS. For 70 of these patients, exons and exon-flanking intron sequences of SCN9A, SCN10A, and SCN11A genes were analyzed by Sanger sequencing to validate MIPs-NGS and TSCA-NGS methods [23 (link)]. Local Medical Ethical Committees of Fondazione IRCCS Istituto Neurologico "Carlo Besta" (Italy), Maastricht University Medical Center (the Netherlands), University of Manchester (United Kingdom) and the Deutsche Diabetes Forschungsgesellschaft EV (Germany) approved this study. Informed consent for genetic testing was given by patients to participate in this study.
Genomic DNA was extracted from peripheral blood by using QIAamp DNA Blood Maxi Kit, Puregene^® Blood Core Kit (Qiagen, Hilden, Germany) or NucleoSpin^®8 Blood Isolation kit (Macherey-Nagel, Düren, Germany). Quality and concentration of the DNA was determined by NanoDrop (Thermo Scientific, Wilmington, USA), and Qubit^® 2.0 Fluorometer using the Qubit^® dsDNA BR assay kit (Life technologies, Bleiswijk, The Netherlands). Isolated DNA was stored with a unique numeric code in the central DNA bank at Maastricht University Medical Centre and IRCCS Foundation “Carlo Besta” Neurological Institute.

Genomic DNA was isolated from whole blood samples using a Puregene Blood Core kit (Qiagen, Valencia, California, USA). We genotyped five TYK2 SNPs—including four previously described as associated with SLE (rs280500, rs12720270, rs2304256, and rs12720356) and the rare functional variant rs34536443—using TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA). The results were analyzed using SDS 2.3 software (Applied Biosystems). All SNPs had a call rate of over 98%. To validate the SNP genotyping accuracy, we directly sequenced samples from a randomly selected subset of 15 patients and 15 controls using an automated ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Life Technologies, CA, USA). All sequences showed 100% reproducibility. Ancestry was determined in the discovery cohort using 96 ancestry informative markers (AIMs) contained in a GoldenGate Genotyping Assay (Illumina, San Diego, CA), as previously described^{29 (link)}. Allele frequencies from the continental populations were obtained from the 1000 Genomes Project Database^{30 (link)}.

DNA extraction from fingernails, peripheral blood lymphocytes, and liver tissue was performed using the DNA extraction DNA Extractor FM Kit (WAKO), Puregene Blood Core Kit (QIAGEN), and QIAamp DNA Mini (QIAGEN), respectively. DNA samples were quantified using a Qubit fluorometer (Life Technologies, Carlsbad, CA, USA) and a NanoDrop system (Thermo Fisher, Waltham, MA). Moreover, quality control of each sample was performed using an Agilent 2200 TapeStation System (Agilent Technologies, Santa Clara, CA, USA). First, 29-845 ng of genomic DNA was fragmented to an average length of approximately 150 bp using a Covaris S220 sonication device. The fragments were purified, end-repaired, A-tailed, and ligated to adaptors. The libraries were constructed by the KAPA HyperPlus Library Preparation Kit (Kapa Biosystems, Wilmington, MA, USA) or NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA, USA). Quality control of the libraries was performed using an Agilent 2200 TapeStation System (Agilent Technologies, Santa Clara, CA, USA).

Umbilical cord blood, umbilical cord tissue, or saliva was obtained from the study subjects. Commercial kits were used to extract genomic DNA from blood (UltraClean Blood DNA Isolation Kit; MO BIO Laboratories, Inc., Carlsbad, CA, USA or Puregene Blood Core Kit; Qiagen, Hilden, Germany) and cord tissue (Gentra Puregene Tissue Kit, Qiagen). OraGene DNA collection kits (DNA Genotek, Ontario, Canada) were used for collecting saliva, and DNA was extracted with the prepIT-L2P kit (DNA Genotek). Genome-wide SNP genotyping was performed with the Infinium HumanCoreExome BeadChip (Illumina, San Diego, CA, USA) by the Technology Centre, Institute for Molecular Medicine Finland (FIMM), University of Helsinki.