End-point genotyping of APOE Single Nucleotide Polymorphisms (SNPs) rs429358 [C/T] and rs7412 [C/T] was performed using TaqMan and OpenArray technologies (Life Technologies, Carlsbad, USA). Genomic DNA was introduced to nanoliter reaction wells on a high-density microplate containing validated TaqMan primers and probes. PCR amplification was completed as recommended by the manufacturer. Water non-template and known positive controls were plated on each array for internal quality assurance and quality checks. Arrays were imaged after amplification on one of two OpenArray NT imagers. SNP genotypes were ascribed after clustering VIC and FAM signals (STATA 12.1, College Station, TX), and were then used to determine the ApoE alleles present(Zuo, van Dyck et al. 2006 (link)).
Openarray
Manufactured by Thermo Fisher Scientific
Sourced in United States
The OpenArray is a high-throughput real-time PCR platform designed for gene expression analysis, genotyping, and other molecular biology applications. It enables simultaneous processing of up to 3,072 samples and assays in a single run. The OpenArray provides a rapid and efficient way to perform targeted, multiplexed nucleic acid analysis.
Automatically generated - may contain errors
Lab products found in correlation
Taqman snp technology, by Thermo Fisher Scientific (7 mentions)
Ready to use human assays library, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Stata 12, by StataCorp (1 mentions)
Mirneasy and rneasy cleanup kits, by Qiagen (1 mentions)
Openarray system, by Thermo Fisher Scientific (1 mentions)
Taqman open array genotyping system, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna midi kit, by Qiagen (1 mentions)
Freedom evo 150 robotic workstation, by Tecan (1 mentions)
Magna pure 96 dna and viral na small volume kit, by Roche (1 mentions)
Axiom analysis suite 3, by Thermo Fisher Scientific (1 mentions)
Ribopure kit, by Thermo Fisher Scientific (1 mentions)
Quantstudio 12k flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Open array plate, by Thermo Fisher Scientific (1 mentions)
Quantstudio 12k flex openarray accufill system, by Thermo Fisher Scientific (1 mentions)
17 protocols using openarray
1
Genotyping APOE SNPs rs429358 and rs7412
2
Genotyping of PROX1 SNP
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DNA was extracted from the peripheral blood leukocytes using a classical salting out method. All SNPs were genotyped by TaqMan SNP technology from ready to use human assays library (Applied Biosystems, USA) using a high throughput genotyping system—OpenArray from Life Technologies (USA). SNPs analysis was performed in duplicate, following the manufacturer’s instructions. As a negative control, we used a sample without template. The negative control was helpful in measuring any false positive signal caused by contamination. No significant deviation from Hardy–Weinberg equilibrium was observed for the studied rs340874 SNP in PROX1 (p > 0.05).
3
Genotyping of FTO Variants
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We selected and genotyped 6 previously identified FTO SNPs in rs3751812 (G > T), rs8050136 (A > C), rs9939609 (T > A), rs6499640 (G > A), rs8044769 (C > T), and rs7190492 (A > G), based on the validated catalog of published genome-wide association studies [16 (link)]. DNA was extracted from peripheral blood leukocytes using a classical salting-out method. The SNPs were genotyped with TaqMan SNP technology from a ready-to-use human assay library (Applied Biosystems, Beverly, MA, USA) using a high-throughput genotyping system, OpenArray (Life Technologies, South San Francisco, CA, USA). SNP analysis was performed in duplicate, following the manufacturer’s instructions. As a negative control, we used a sample without a template. The negative control was applicable in measuring any false positive signal caused by contamination.
4
Genotyping of Blood DNA SNPs
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DNA was extracted from the peripheral blood leukocytes using a classical salting out method. All SNPs were genotyped by TaqMan SNP technology from ready to use human assays library (Applied Biosystems, USA) using a high throughput genotyping system—OpenArray from Life Technologies (USA). SNPs analysis was performed in duplicate following the manufacturer’s instructions. As a negative control, we used a sample without template. The negative control was helpful for measuring any false positive signal caused by contamination. No significant deviation from Hardy Weinberg equilibrium was observed for either of the SNPs in this study (all p > 0.05).
5
Extracellular RNA Quantification Protocol
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Five ml of cleared secretome was 1:1 diluted in PBS before ultracentrifugation at 100,000× g for 9 h at 4 °C. Resulting pellets were processed with miRNeasy and RNeasy Cleanup Kits (Qiagen, Hilden, Germany) after addition of 30 pg of exogenous Arabidopsis thaliana ath-miR-159a synthetic miRNA spike. This was used to evaluate RNA recovery and cDNA synthesis performed as previously reported [14 (link)]. The OpenArray system (Life Technologies, Foster City, CA, USA) was used to determine miRNA expression in 384-well OpenArray plates according to the manufacturer’s protocol. Each single miRNA was considered as present when amplification resulted in all three samples. Eventually, ath-miR-159 spike-in CRT was used to equalize technical differences, and the global mean method [15 ] allowed normalization between samples.
6
Genotyping Skin Color SNPs in VDR
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SNPs within the VDR gene and within skin color genes (TYR, TYRP1, OCA2, SLC45A2, SLC24A5, KITLG and MC1R) were selected to fill a 64-SNP array as indicated in Supplemental Material, Table S1 . Genotyping was performed at a central laboratory (L.A. and M.F.-C.) using Applied Biosystems Taqman and OpenArray technologies (Life Technologies, Carlsbad). SNPs were typed using TaqMan genotyping chemistry supported on a metal-based array. DNA samples were loaded and amplified on arrays as recommended by the manufacturer. Arrays were scanned on the OpenArray NT imager and genotypes were called using the OpenArray SNP Genotyping analysis software. All of the eight loci we surveyed were on different chromosomes; so interlocus was the same as interchromosomal in all of our analyses.
7
DNA Extraction and SNP Genotyping
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DNA was extracted from the peripheral blood leukocytes using a classical salting out method. The SNPs were genotyped with TaqMan SNP technology from a ready-to-use human assay library (Applied Biosystems, MA, USA) using a high-throughput genotyping system, OpenArray (Life Technologies, CA, USA). A sample without a template was used as a negative control. The negative control was applicable in measuring any false positive signals caused by contamination.
8
Genotyping of Common FTO SNPs
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We genotyped 4 common FTO SNPs in rs3751812 (G > T), rs8044769 (C > T), rs8050136 (A > C), and rs9939609 (T > A). DNA was extracted from peripheral blood leukocytes using a classical salting-out method. The SNPs were genotyped with TaqMan SNP technology from a ready-to-use human assay library (Applied Biosystems, MA, USA) using a high-throughput genotyping system, OpenArray (Life Technologies, CA, USA). SNP analysis was performed in duplicate, following the manufacturer’s instructions. We used a sample without template as a negative control to detect possible false positive signals caused by contamination.
9
MC4R SNP Genotyping from Blood Leukocytes
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We genotyped previously identified MC4R SNPs in: rs17782313, rs12970134, rs633265, and rs1350341. DNA was extracted from peripheral blood leukocytes using a classical salting out method. The SNPs were genotyped with TaqMan SNP technology from ready-to-use human assays library (Applied Biosystems, Foster City, CA, USA) using a high throughput genotyping system (OpenArray, Life Technologies, Waltham, MA, USA). SNP analysis was performed in duplicate according to the manufacturer’s instructions. To detect possible false positive signals caused by contamination, a negative control consisting of a sample without a template was used.
10
Genotyping BDNF Variants for Obesity Risk
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Based on the available scientific literature26 (link), we selected and genotyped 4 BDNF polymorphisms that are considered genetic risk factors for obesity: rs6265, rs4923461, rs10501087, and rs10835211. DNA was extracted from the peripheral blood leukocytes by a classical salting-out method. The analyzed SNPs were genotyped with a TaqMan SNP technology from a ready-to-use human assay library (Applied Biosystems, MA, USA) using a high-throughput system (Open Array, Life Technologies, CA, USA). We performed SNP analysis in duplicate, according to the manufacturer’s instructions. As a negative control we used a sample without a template to detect possibly caused by contamination false-positive signals.
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