Lightcycler 480 sw

Quantitative PCR was performed using the LightCycler 480 II (Roche), according to manufacturer’s instructions, using a mixture containing 45 ng cDNA, 1xTaqMan Universal PCR Master Mix, no AmpErase UNG (Applied Biosystems), 1xTaqMan SNP Genotyping Assay (Applied Biosystems), and nuclease-free water (Ambion) in a 20 μl reaction volume. ACTB (Applied Biosystems, cat#Hs99999903_m1) was included as reference gene. A standard curve was generated using pooled equal amounts of cDNA from all samples. Quantification of the dual-color hydrolysis of both allele-specific fluorescent reporter dyes, FAM (“G” allele) and VIC (“A” allele), was performed with the LightCycler 480 SW 1.5.1 software (Roche) using the 2^nd derivative method, according to manufacturer’s instructions.

Total genomic DNA was extracted from blood leukocytes with a QIAamp DNA Blood Mini or Maxi Kit (Qiagen, Tokyo, Japan). Genotyping of rs7832552, rs6552828, and rs1800795 was performed at the Institute of Health and Sports Science and Medicine (Juntendo University) with Real Time Thermocycler (LightCycler 480, Roche Applied Science, Mannheim, Germany) using TaqMan SNP genotyping assay method. PCR 384-well plates were read on LightCycler 480 using the end-point analysis mode. Allelic discrimination analysis was performed with a LightCycler 480 SW software version 1.5.1.62 (Roche Applied Science, Mannheim, Germany).

Total DNA was isolated from the saliva of all participants in both Study 1 and 2 with an Oragene® DNA Collection Kit (DNA Genotek, ON, Canada) and quantified using a NanoDrop 8000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, DE, USA) or Eppendorf Bio Photometer Plus (Eppendorf, Tokyo, Japan). DNA samples were stored at 4 °C until use. The samples were analyzed for the rs12722 polymorphism in the 3′-UTR of COL5A1 using a TaqMan SNP Genotyping Assay (Assay ID: C____370252_20) and LightCycler® 480 System (Roche Molecular Systems, Mannheim, Germany) or StepOne™ Real-Time PCR System (Thermo Fisher Scientific). Five microliters of the genotyping mixture contained 2.5 μL TaqMan® GTXpressTM Master Mix (2×) or TaqMan® Universal Master Mix II (2×), 0.0625 μL TaqMan® SNP Genotyping Assay mix (40×), 1.4375 μL sterilized water, and 1 μL genomic DNA (10 ng/μL). Two to four negative controls were included on each plate. Genotypes were called based on TaqMan® assays results using LightCycler® 480 SW (version 1.5, Roche Molecular Systems) or StepOne™ software (version 2.3, Thermo Fisher Scientific). Three hundred eighty randomly selected samples were genotyped in duplicate for the rs12722 polymorphism, and we confirmed that the genotyping results perfectly agreed between duplicates.