Assay loading reagent

Biomark^™ HD with a 96.96 IFC was used for the RT-qPCR amplification, as previously described [17 (link)]. Briefly, for each sample, a 6 µL sample mix containing 3 µL of 2× SsoFast EvaGreen Supermix with low ROX (Bio-Rad Laboratories, Hercules, CA, USA), 0.3 µL of 20× DNA Binding Dye (Fluidigm), and 2.7 µL of the pre-amplified sample was prepared. A primer stock (100 μM combined forward and reverse primers) was prepared for each assay, and 0.3 µL of the stock was mixed with 3 µL of 2× Assay Loading Reagent (Fluidigm) and 2.7 µL 1× DNA Suspension Buffer to make assay mixes. Finally, 5 μL of each assay and sample mix was transferred into the appropriate inlets according to Fluidigm’s recommendation. After loading, the array was placed in the Biomark HD instrument for quantification and detection using the GE Fast 96 × 96 PCR + Melt v2.pcl PCR thermal protocol. The data were analyzed with Real-Time PCR Analysis Software (Fluidigm) according to Fluidigm’s recommendation. A non-template cDNA/pre-amplification and a non-template pre-amplification control (H₂O) were included and were finally defined as those with Ct values ≥ 35 or that were undetermined.

From these 46 SNPs, we searched our GWAS of COME/ROM data to determine if any associations had already tested. To investigate the remaining SNPs from the Raine GWAS results, we genotyped the SNPs in the UMN family population using the Fluidigm SNPtype assay platform (http://www.fluidigm.com/snptype-assays.html). Briefly, 100 ng/uL of each DNA sample was combined with Biotium 2× Fast Probe Master Mix, SNPtype Sample Loading Reagent (Fluidigm), and the reference dye ROX (Invitrogen Inc.). Each SNP type assay was mixed with 2× Assay Loading Reagent (Fluidigm). Both sample mixes and assay mixes were then loaded onto Fluidigm 96.96 Dynamic Genotyping Arrays, and nanofluidic circuitry loads and mixes the 96 loci with 96 samples in 9216 reaction chambers. Thermocycling was performed on Fluidigm's Stand-alone Thermal Cycler and fluorescence detection performed on the EP1 genotyping system (Fluidigm).
SNP rs2704219, which did not pass quality control measures, was re-genotyped after a pre-amplification step performed with the following thermocycling conditions: 95°C for 15 minutes, then 14 cycles of 5 seconds at 95°C and 4 minutes at 60°C. Pre-amplified DNA was then diluted 1∶100 in suspension buffer and genotyped as previously described.

Each sample was pre-amplified 18 cycles with a mix of 15 primer pairs (without 18 S). The reaction contained 10 μl of iQ Supermix (Bio-Rad), 4 μl of cDNA, 2 μl of pooled primers with a final concentration of each primer of 25 nM and 4 μl of water. Temperature profile was 95 °C for 15 s and 60 °C for 4 min. As a control, NTC was included in the pre-amplification reaction, one extra sample was included as IPC. The pre-amplified cDNA was immediately used or placed in freezer at −20 °C. The pre-amplified cDNA was diluted 10x with water prior to the use. qPCR was performed using the high-throughput platform BioMark™ HD System (Fluidigm) and two 48.48 GE Dynamic Arrays. Five μL of sample pre-mix contained 1 μL of 10× diluted pre-amplified cDNA, 2.5 μL of Taqman universal mastermix II without UNG (Applied Biosystems), 0.25 μL of 20× GE sample loading reagent (Fluidigm) and 1.25 μL of water. Five μL of assay pre-mix contained 1.25 μL of 12 μM primer/probe assays with PerfectProbeTM (Primer Design) with final concentration of 300 nM in reaction, 2.5 μL of 2× assay loading reagent (Fluidigm). Thermal conditions for qPCR were: 95 °C for 10 min, 35 cycles of 95 °C for 15 s and 60 °C for 60 s.