The single-nucleotide polymorphisms (SNPs) in the LDLR (rs688, rs5925), APOB (rs676210, rs1042031, and rs676210), ABCC8 (rs6544718 and rs6544713), LPL (rs285, rs3735964, and rs13702), HNF1A (rs2650000), ApoE (rs429358 and rs7412), and HNF4A (rs1800961) genes, selected for this study, were genotyped by Real-Time Polymerase Chain Reaction (PCR) using TaqMan® (Thermo Fischer Scientific, Foster City, CA, EUA) systems. The amplification was performed in a final volume of 12.5 μL, containing 0.63 μL of the specific TaqMan assay (Table 1 ) for genotyping each referred SNPs (including forward and reverse primers and probes marked with VIC and FAM), 6.25 μL of TaqMan® Genotyping Master Mix (2X TaqMan®Genotyping Master Mix, Thermo Fisher Scientific), 20 ng of genomic DNA, and DNAse/RNAse free water in enough quantity to complete the final genotyping reaction. The following conditions were used for cycling the SNPs on a qPCR thermocycler (StepOnePlus Real-Time PCR System, Thermo Fisher Scientific): 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 1 min. The genotyping analysis was performed with an online software (Applied Biosystems™ Analysis Software, Genotyping Analysis Module, version 3.2.).
Taqman genotyping master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Italy
TaqMan Genotyping Master Mix is a reagent used in polymerase chain reaction (PCR) assays for genotyping. It contains all the necessary components, including DNA polymerase, for performing real-time PCR experiments.
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Lab products found in correlation
Taqman snp genotyping assay, by Thermo Fisher Scientific (88 mentions)
Taqman probe, by Thermo Fisher Scientific (24 mentions)
Qiaamp dna blood mini kit, by Qiagen (21 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (20 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (19 mentions)
Taqman genotyping assay, by Thermo Fisher Scientific (17 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (17 mentions)
Qiaamp dna mini kit, by Qiagen (14 mentions)
Taqman assay, by Thermo Fisher Scientific (13 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (11 mentions)
Sds 2, by Thermo Fisher Scientific (10 mentions)
Taqman genotyper software, by Thermo Fisher Scientific (9 mentions)
Custom taqman snp genotyping assay, by Thermo Fisher Scientific (9 mentions)
Taqman copy number assay, by Thermo Fisher Scientific (9 mentions)
Quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (8 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (8 mentions)
Steponeplus, by Thermo Fisher Scientific (8 mentions)
Copycaller software, by Thermo Fisher Scientific (8 mentions)
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Taqman, by Thermo Fisher Scientific (7 mentions)
427 protocols using taqman genotyping master mix
1
Genetic Profiling of Dyslipidemia Markers
2
High-Throughput Genotyping via TaqMan PCR
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Genotyping was performed using TaqMan-based real-time PCR with TaqMan assays from Applied Biosystems™ (Table 1 ). Pre-coated 384-well TaqMan® Custom Plating (Applied Biosystems™, P/N: 4462783) which contains 32 pharmacogenomics assays was used. Up to 11 samples plus one no-template control were included in each run. A final volume of 5 μl consisting of 2.5 μl TaqMan® Genotyping Master Mix (Applied Biosystems™, P/N: 4371355) and 2.5 μl genomic DNA was dispensed to each well by epMotion M5073 automated pipetting system (Eppendorf). The TaqMan® Genotyping Master Mix (Applied Biosystems™, P/N: 4371355) contains ROX dye which serves as a passive reference dye to normalize signal and ensure data integrity. The qPCR was run on QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems™) with the following thermal cycling condition: pre-PCR hold at 60°C for 30 seconds, initiation at 95°C for 10 minutes for initial denaturation and enzyme activation, followed by 50 cycles of 95°C for 15 seconds and 60°C for 90 seconds.
3
Genetic Polymorphisms Analysis in FFPE Samples
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DNA was extracted from the FFPE samples. Applying micro-dissection on 10 µm sections, non-tumor tissues were dissected from the FFPE samples. DNA extraction was performed using a QIAamp DNA FFPE Tissue kit (Qiagen GmbH) according to the manufacturer's protocol. The TaqMan technique was then used to determine FcγR2A-H131R rs1801274, FcγR3A-V158F rs396991 and EGFR-R521K rs2227983 polymorphisms using established primers (22 (link)), TaqMan SNP Genotyping Assays C—9077561_20, C—25815666_10 and C—16170352_20 (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the TaqMan Genotyping Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). In brief, a 5-µl reaction solution, containing TaqMan Genotyping Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), Assay Mix, and 20–40 ng of genomic DNA diluted in dH2O, was incubated in 384-well microtiter plates at 50°C for 2 min to degrade dU-containing DNA, followed by incubation at 95°C for 10 min (denaturation), followed by 40 cycles of 15 sec at 95°C and 1 min of annealing and extension at 60°C. The ABI Prism 7900HT (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for end-point reading of the fluorescence generated during PCR amplification. EGFR CA Repeats in Intron 1 Genotyping was determined via direct sequencing, as previously described (23 (link),24 (link)).
4
Copy Number Variation Analysis of FGFR4 in ccRCC
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After thawing each ccRCC specimen preserved in RNA at ˗80 °C, DNA extraction was performed according to the recommended protocol using the Easy DNA Extraction Kit® (KANEKA, Tokyo, Japan). DNA concentration was measured using the NanoDrop Lite spectrometer® (Thermo Fisher Scientific, Waltham, MA, USA). The DNA concentration of each specimen was adjusted to 5 ng/µL using deuterium depleted water. The PCR components included the TaqMan Copy Number Assays® (ID: Hs01973966_cn, Life Technologies, Carlsbad, CA, USA) for FGFR4 detection, the TaqMan RNaseP control reagent® (Life Technologies) as an endogenous control, and the TaqMan Genotyping Master Mix® (Life Technologies) as the substrate. Multiplex PCR mixtures, of total volume 20 µL, contained the TaqMan Genotyping Master Mix (Life Technologies), RNaseP Primer-Probe (VIC dye) Mix, FGFR4 Primer-Probe Mix (FAM dye), and 5 ng of genomic DNA. All experiments were conducted in triplicate. The PCRs were performed on the ABI7300 Fast Real-Time PCR System with TaqMan detector® (Life Technologies), and data were analysed using Copy Caller software version 2.1® (Thermo Fisher Scientific). Detection of 2.5 or more copies of the gene indicated copy number amplification.
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After thawing each ccRCC specimen preserved in RNA at ˗80 °C, DNA extraction was performed according to the recommended protocol using the Easy DNA Extraction Kit® (KANEKA, Tokyo, Japan). DNA concentration was measured using the NanoDrop Lite spectrometer® (Thermo Fisher Scientific, Waltham, MA, USA). The DNA concentration of each specimen was adjusted to 5 ng/µL using deuterium depleted water. The PCR components included the TaqMan Copy Number Assays® (ID: Hs01973966_cn, Life Technologies, Carlsbad, CA, USA) for FGFR4 detection, the TaqMan RNaseP control reagent® (Life Technologies) as an endogenous control, and the TaqMan Genotyping Master Mix® (Life Technologies) as the substrate. Multiplex PCR mixtures, of total volume 20 µL, contained the TaqMan Genotyping Master Mix (Life Technologies), RNaseP Primer-Probe (VIC dye) Mix, FGFR4 Primer-Probe Mix (FAM dye), and 5 ng of genomic DNA. All experiments were conducted in triplicate. The PCRs were performed on the ABI7300 Fast Real-Time PCR System with TaqMan detector® (Life Technologies), and data were analysed using Copy Caller software version 2.1® (Thermo Fisher Scientific). Detection of 2.5 or more copies of the gene indicated copy number amplification.
5
Genomic DNA Extraction and SNP Genotyping
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DNA was isolated from whole blood by anion exchange membrane column separation (Genomic Maxi AX Blood; A&A Biotechnology, Gdynia, Poland) as per the manufacturer's protocol. Initial screening of DNA purity was determined based on the evaluation of optical density ratio at 260/280 nm (samples with a ratio of 1.8–2.0 were considered to be high purity).
SNPs (rs4977574 and rs7857345 located in the CDKN2B-AS1 gene, and rs3798220 and rs10455872 located in the LPA gene) were analyzed with allelic discrimination technique. The analysis was performed on 384-well plates (Applied Biosystems, Foster City, CA, USA) using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Reactions were performed in the final volume of 5.1 μl per sample containing 0.125 μl of 40 × TaqMan® SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA), 2.5 μl of 2 × TaqMan Genotyping Master Mix (Applied Biosystems, Foster City, CA, USA), and 2.5 μl of DNA (at concentrations of 3 ng/μl).
SNPs (rs4977574 and rs7857345 located in the CDKN2B-AS1 gene, and rs3798220 and rs10455872 located in the LPA gene) were analyzed with allelic discrimination technique. The analysis was performed on 384-well plates (Applied Biosystems, Foster City, CA, USA) using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Reactions were performed in the final volume of 5.1 μl per sample containing 0.125 μl of 40 × TaqMan® SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA), 2.5 μl of 2 × TaqMan Genotyping Master Mix (Applied Biosystems, Foster City, CA, USA), and 2.5 μl of DNA (at concentrations of 3 ng/μl).
6
Genotyping of TNFA gene variants
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Blood samples were collected from all subjects of the study. The samples were kept at 4 °C until DNA extraction, which was performed within 6 h of blood collection. DNA was extracted from peripheral leucocytes using DNAzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Four SNPs located in the promotor of TNFA gene were selected, namely rs361525, rs1800629, rs1799964, and rs1799724. SNPs were genotyped with commercially available TaqMan allelic discrimination assays (Life Technologies) using the 7900 HT Real-time PCR System (Applied Biosystems, Foster City, CA, USA). A mix of unlabelled PCR primers and TaqMan MGB probes labelled with FAM or VIC dye were used. The reaction was performed in a 10-μL solution that contained 0.5 μL of a 40× oligonucleotides mix, 5 μL of a 2 × TaqMan Genotyping Master mix (Applied Biosystems, all), and 2 μL of 50 ng genomic DNA. PCR conditions were as follows: initial denaturation at 95 °C for 15 min; 40 cycles at 95 °C for 10 s and 60 °C for 45 s. Genotyping was performed on coded and blinded samples in the presence of negative ones (no DNA) included in each 96-well plate for quality control. The genotyping results were determined by using SDS 2.3 Allelic Discrimination Software (Applied Biosystems).
7
Allele-Specific qPCR for KRAS and BRAF
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For single-gene assays, allele-specific qPCR technology was performed using TaqMan probes for KRAS (p.Gly12Ser/Cys/Asp/Ala/Val and p.Gly13Asp) and for BRAF p.Val600Glu (Thermo Fisher Scientific, in-house design, available on request, Waltham, MA, USA).
Each real-time quantitative PCR, one per probe, was run in a final volume of 5 µL in 384-well plates, including 2.5 µL of 2× TaqMan genotyping master mix (Applied Biosystems, Foster City, CA, USA), 0.5 µL of 10× Assay Mix, 1 µL of deionized water, and 10 ng of DNA template. Samples were run in duplicate on an ABI Prism 7900 HT sequence detection system (Applied Biosystems) using standard thermocycling conditions and analyzed with SDS software version 2.4 (Applied Biosystems). KRAS and BRAF mutation status was determined according to the manufacturer’s instructions and the results obtained for these 2 genes, in the hotspots defined above, were combined, and compared with those obtained in NGS to define the mutation status of the patients.
Each real-time quantitative PCR, one per probe, was run in a final volume of 5 µL in 384-well plates, including 2.5 µL of 2× TaqMan genotyping master mix (Applied Biosystems, Foster City, CA, USA), 0.5 µL of 10× Assay Mix, 1 µL of deionized water, and 10 ng of DNA template. Samples were run in duplicate on an ABI Prism 7900 HT sequence detection system (Applied Biosystems) using standard thermocycling conditions and analyzed with SDS software version 2.4 (Applied Biosystems). KRAS and BRAF mutation status was determined according to the manufacturer’s instructions and the results obtained for these 2 genes, in the hotspots defined above, were combined, and compared with those obtained in NGS to define the mutation status of the patients.
8
Genotyping of NOTCH Receptors SNPs
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A total of four single nucleotide polymorphism in NOTCH1, NOTCH2, NOTCH3 and NOTCH4 receptor genes were selected from previous literature [18 (link)–24 (link)]. TaqMan allelic discrimination assays were used to genotype the SNPs based on Livak’s method [25 (link)]. Briefly, for each sample, 20 ng of purified genomic DNA was mixed with 5.0 µl of 2× TaqMan genotyping Master Mix (Catalog no. 4371355, Applied Biosystems, Foster City, CA, United States) and 0.25 µl of 40× TaqMan SNP genotyping assay (Catalog no. 4351379, Assay ID: C____189,059_10; C__31617470_30; C___7494157_10; C__27523194_10, Thermo Fisher Scientific, United States) containing the primers and probe in a total volume of 10 µL performed in Fast Optical 96-Well Reaction Plate (Catalog no. 4346906, Applied Biosystems, Foster City, CA, United States). The genotypes were determined by endpoint reading on QuantStudio 7 Flex Real Time PCR system (Applied Biosystems, Foster City, CA, United States). The instrument was programmed as follows: pre-read at 60°C for 30 s, polymerase activation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min followed by post-read at 60°C for 30 s. TaqMan Genotyper Software version 1.4 was used to automatically analyze the data and make the genotype calls.
9
Genotyping Four Key SNPs in Peripheral Blood
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Genomic DNA was obtained from peripheral blood withdrawn on EDTA, using commercially available kits (Quick gDNA MiniPrep kit, Zymo Research, USA; PureLink Genomic DNA Mini Kit, Invitrogen, Thermo Fisher, USA). We genotyped four SNPs (FDPS rs2297480, LRP5 rs3736228, SOST rs1234612, VKORC1 rs9934438) in all patients and controls using the real-time PCR technique. Standard, predesigned TaqMan SNP genotyping assays, containing all the primers and probes needed for genotyping, were purchased from Thermo Fisher (codes C___2737970_10, C__25752205_10, C___7566033_10, C__30204875_10). All the genotyping were performed according to manufacturer’s instructions. The reaction mix contained 10 μl of 2xTaqMan Genotyping Master Mix (Applied Biosystems, Thermo Fisher, USA), 0.5 μl of the corresponding 40xTaqMan SNP genotyping assay, approximately 25 ng of genomic DNA and free-nucleases water to the final volume of 20 μl. The same amplification program was used for all the genotyping, consisting in a pre-read stage of 30 seconds (s) at 60°C, hold stage of 10 minutes (min) at 95°C, followed by the PCR stage, consisting of 40 cycles, each comprising 15 s at 95°C and 1 min at 60°C, and a post-read stage of 30 s at 60°C. All the experiments were run on a QuantStudio 3 real-time PCR machine (Applied Biosystems, Thermo Fisher, USA).
10
COMT Genotyping Using TaqMan Assay
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The rs4680 polymorphism of COMT was assayed using a predesigned TaqMan Genotyping Assay (Assay ID: C__25746809_50; Applied Biosystems, USA). Amplification was performed using manufacturer recommended conditions for a 96-well plate. Dilute samples of 5 ng of DNA in 11.25 μL dH2O were mixed with 12.5μL of 2X TaqMan® Genotyping Master Mix (Applied Biosystems, USA) and 1.25μL of 20X TaqMan® Drug Metabolism Genotyping Assay Mix (Applied Biosystems, USA) for a total reaction volume of 25μL. The cycling profile was: 95°C for 10 min followed by 50 cycles of 92°C for 15 sec and 60°C for 90 sec. Genotypes were determined after amplification using dye fluorescence intensities via the Bio-Rad CFX Manager with lab member and blank controls in triplicate as references. Individuals were coded as Val/Val homozygotes, Val/Met heterozygotes, and Met/Met homozygotes for analysis.
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