Nucleon bacc3 kit

DNA was extracted from blood using the Nucleon BACC3 Kit (GE Healthcare) and from saliva using the Oragene DNA Kit (DNA Genotek) according to the manufacturers’ directions. FFPE tissue blocks were sectioned to 8 μm, dewaxed and rehydrated using a standard xylene-ethanol deparaffinisation protocol. Tumour and benign glands were marked on haematoxylin and eosin stained tissue sections by a pathologist (SD, RM). Marked tumour and benign regions were macro-dissected separately for both DNA and RNA. DNA was extracted using the QIAamp DNA FFPE Tissue Kit (QIAGEN) according to the manufacturer’s instructions and eluted in 50 μL of ATE Buffer. DNA was quantified using the Nanodrop® ND-1000 UV-vis spectrophotometer (Nanodrop® Technologies). RNA was extracted using the RecoverAll Total Nucleic Acid Isolation Kit (ThermoFisher Scientific) according to the manufacturer’s instructions and eluted in 30  μL of dH₂O. RNA quality and quantity was assessed using the 2100 Bioanalzyer (Agilent). The SuperScript^TM VILO^TM cDNA Synthesis Kit (Invitrogen) was used for cDNA conversion, as per the manufacturer’s instructions.

Genomic DNAs from both peripheral blood samples with EDTA-K2 (S1, S2, S3) and buccal cells (S4, S5) were extracted using the Nucleon BACC3 kit (GE Healthcare, Hatfield, UK) and Puregene Buccal Core Kit A (Qiagen Science, Germantown, MD, USA), respectively. Genomic DNAs from fresh tissue, tissue from the slide, and tissue from FFPE (TS1, TS2, and TS3) were purified by Kit Qiamp fast Tissue and Qiamp DNA FFPE tissue Kit (Qiagen Science, USA). Before proceeding to library preparation, the concentration and purity of the DNAs were assessed both by fluorimetric method, using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and by a spectrophotometric method by Nanodrop instrument (Thermo Fisher Scientific). The DNAs integrity was evaluated by ScreenTape analysis on TapeStation (Agilent, Santa Clara, CA, USA).

Genomic DNA was extracted from 2 ml EDTA-blood of RRMS patients using the Nucleon BACC3 kit (GE-Healthcare) according to the manufacturer’s instructions. Briefly, blood cells were lysed at RT, and pellet was stored at −80°C. Precipitated DNA was airdried for 10 min, resuspended in DNAse/RNAse free water overnight at 4°C and quantified using Qubit dsDNA HS Assay Kit and Qubit 4 Fluorometer (Invitrogen). Class II HLA-DQA1, DQB1, DRB1, and DRB3/4/5 genotypes were determined by single molecule real time sequencing of exons 2–6 and introns 2–5 at 8× resolution (Histogenetics, USA). Genotypes were converted to serotypes according to the 2010 nomenclature of HLA system (46 (link)). HLA-DRB1, -DQA1 and -DQB1 typing data of 1,000 healthy donors were a resource of the Milieu Interieur consortium. Alleles were imputed at four-digit resolution from the analysis of 5,699,237 SNPs using SNP2HLA v1.0370 (47 (link)).

Four functionally-relevant TLR2 and TLR4 single-nucleotide polymorphisms (SNPs) (rs4696480, rs3804099 and rs1927914, rs11536891 respectively) were selected based on previous genetic association studies of BD [22 (link),23 (link)]. Genomic DNA was extracted from EDTA-treated peripheral blood samples or B-lymphoblastoid cell lines using the Nucleon BACC3 kit (GE HealthCare, Chalfont St Giles, UK). The four SNPs (TLR2 intron 1 rs4696480 A/T, TLR2 exon 3 rs3804099 C/T and TLR4 promoter rs1927914 A/G and 3’UTR rs11536891 C/T) were analyzed using pre-developed TaqMan 5’-nuclease assay kits (Applied Biosystems, Foster City, CA, USA) with allele-specific fluorogenic oligonucleotide probes (C__27994607_10, C__22274563_10, C__2704048_10 and C__31784036_10, respectively), as previously reported.

Genomic DNA was extracted from EDTA-treated peripheral blood samples or B-lymphoblastoid cell lines using the Nucleon BACC3 kit (GE HealthCare, Chalfont St Giles, UK). The genotyping of the two SNPs herein studied (intron 1 rs16910631G/A and intron 3 rs2078178 A/G) was performed using a TaqMan 5’-nuclease assay (Applied Biosystems, Foster City, CA, USA) with allele-specific fluorogenic oligonucleotide probes. The following pre-developed TaqMan assay genotyping kits were used: C_33748498_10 and C_1932439_10.

Genomic DNA was extracted from EDTA-treated peripheral blood samples using the Nucleon BACC3 kit (GE HealthCare, Chalfont St Giles, UK). HLA two digits intermediate resolution typing for HLA-DRB1 and HLA–DQB1 alleles was performed using PCR-sequence specific oligonucleotide (SSO) Luminex LABTYPE^®SSO kits designed to recognize all the broad specificities based on the sequence databases from IMGT/HLA Database (database version 3.17.0) (http://www.ebi.ac.uk/ipd/imgt/hla/probe.html). The Luminex 100 flow analyzer identified HLA alleles via HLA visual 1.0 software, by referring to HLA typing template data for the studied loci as provided by the manufacturer (OneLambda, Inc. CA). The detected alleles are assigned into their corresponding serological specificities with consequent HLA typing results as serological equivalents. HLA-DQ2 to -DQ9 will be the corresponding serological specificities for HLA-DQB1 alleles while those corresponding to HLA-DRB1*01:01 to –DRB1*10:01 alleles will be HLA-DR1 to -DR18.

The study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Local Ethics Committee of the Hospices Civils de Lyon. Written informed consent was provided by all parents of the patients enrolled in the study. Genomic DNA was extracted from EDTA-preserved whole blood using Nucleon BACC3 kit (GE healthcare, Chalfont Saint Giles, Buckinghamshire, UK). DNA was analyzed by Sanger sequencing or Massively Parallel sequencing (MPS).
For patients 1 and 2, as CLAH was suspected on clinical and biological data, STAR and CYP11A1 genes were immediately Sanger sequenced. For patients 3 and 4 who presented with glucocorticoid deficiency, MC2R and MRAP genes were first Sanger sequenced. DNA was further analyzed by MPS.
Sanger sequencing consisted of selective amplification of the exons and the exon-intron boundaries of the analyzed gene by PCR using specific primers (sequences available upon request), followed by conventional dideoxy sequencing on an ABI-3500XL sequencer (Thermofisher scientific, Watham, MA, USA) and compared to the human genome (GRCh37/hg19 assembly) using SeqScape® software v3 (Thermofisher scientific).
For MPS, a custom panel targeting 57 genes involved in adrenal insufficiency and DSD including the CYP11A1 gene as previously described (22 (link)) was used. Pathogenic mutations found by MPS were verified by Sanger sequencing.