Taqman 5 nuclease assay kit

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 sample using a standard procedure. Selection of analysed single-nucleotide polymorphisms (SNPs) of TLR2 (rs4696480 and rs3804099), TLR4 (rs1927914 and rs11536891) and NOD2 (rs2066842) were based on previous positive genetic association findings with BD subsets (Oliveira et al. 2014a (link), b (link), c (link)). All SNPs were analysed by a pre-developed TaqMan^® 5′-nuclease assay kits (Applied Biosystems^®, Foster City, CA, USA) using allele-specific fluorogenic oligonucleotide probes as described earlier (Oliveira et al. 2014a (link), b (link), c (link)).

Saliva samples were collected using the Oragene DNA self-collection kit (DNAGenotek). DNA was extracted using the Blood & cell culture DNA maxi kit (Qiagen) and concentration evaluated by fluorescence (Qubit). Some 50 nanograms of DNA was used to amplify the region of the BDNF Val66Met polymorphism using a real-time polymerase chained reaction cycler (Ligthcycler 480, Roche) and a TaqMan 5’ nuclease assay kit (Life Technologies). PCR was made using 0.5 ul of 10X PCR MasterMix (Roche), 0.25 ul 40X TaqMan assay, 50 ng DNA in a final volume of 5 uL. PCR run included a denaturing step of 10 min-95°C followed by 35 PCR cycles (1 min 95°C, 1 min 55°C, 1 min 72°C).