The genotype probe of rs224534 came from ABI (USA, 4351379). Using TaqMan 5’-nuclease assay on the TaqMan 7900HT Sequence Detection System (Applied Biosystems, Foster City, California) was under the following conditions: 10 minutes at 95°C (enzyme activation) followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute (annealing/extension). Procedure of the genotyping was consistent with our previous report.[19 (link)] The genotype of failed heart was confirmed by Sanger sequencing with the primers listed in Table S1, http://links.lww.com/MD/G904 .
Taqman 7900ht sequence detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TaqMan 7900HT Sequence Detection System is a real-time PCR instrument designed for sensitive, accurate, and high-throughput gene expression analysis. It utilizes TaqMan technology to detect and quantify specific nucleic acid sequences.
Automatically generated - may contain errors
9 protocols using taqman 7900ht sequence detection system
1
Genotyping Protocol for rs224534
2
Genotyping Human DNA Samples
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DNA of human samples was extracted from peripheral leucocytes according to our previously published method.26 Human peripheral lymphocytes were isolated using lymphocyte separation medium (Tian Jin Hao Yang Biological Manufacture, China, LTS10770125). The SNP rs2070600 was genotyped in our study using the TaqMan 5′‐nuclease assay on the TaqMan 7900HT Sequence Detection System (Applied Biosystems, Foster City, California) under the following conditions: 10 min at 95°C (enzyme activation), 40 cycles at 95°C for 15 s each, and 60°C for 1 min (annealing/extension). The endpoint read was performed for allelic discrimination after amplification. The genotyping procedure was consistent with our previous report.27 Details regarding primers and probes are provided in Supporting Information, TableS2 .
3
Genotyping of RIP3 Variants
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Genomic DNA was extracted from peripheral leucocytes as previously reported.23 ABI Primer Expression 3.0 software was used for design of probe and primer sequences, which were synthesized by Shanghai GeneCore Bio Technologies Co., Ltd, China. The common variants in RIP3 promoter and CDS regions were genotyped using the TaqMan assay on the TaqMan 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) with the following conditions: 10 minutes at 95°C (enzyme activation) followed by 45 cycles at 95°C for 15 seconds and 60°C for 1 minute (annealing/extension). An endpoint read was performed for allelic discrimination after amplification. The details of the procedure for amplification and the quality control of genotyping were mentioned in our previous study.24 Details regarding primers and probes can be found in Table S2 .
4
Genomic DNA Extraction and Genotyping
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We extracted genomic DNA from peripheral blood leukocytes using Tiangen commercially available kit (Tiangen, Beijing, China). Detailed procedures of DNA extraction have been described previously.20 Referring to the Chinese data of the 1000 Genomes, we selected three tagged genetic variants with minor allele frequency (MAF) > 0.05 in the promoter region of MMP2 for further genotyping. The probes for genotyping came from Applied Biosystems (ABI) with the following assay ID: rs243865 (C___3225943_10, 4351379), rs2285052 (C___3225944_10, 4351379), and rs2285053 (C__26734093_20, 4351379). The genotyping procedure was conducted according to the TaqMan assay on the TaqMan 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) with the following conditions: 10 min at 95°C (enzyme activation) followed by 45 cycles at 95°C for 15 s and 60°C for 1 min (annealing/extension). An endpoint read was performed for allelic discrimination after amplification. The details of the procedure for amplification and the quality control of genotyping were mentioned in our previous study.21
5
Genotyping Protocol for Association Analysis
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All subjects were individually genotyped according to the manufacturer’s instructions using the TaqMan 7900HT sequence Detection System (Applied Biosystems, Foster City, Calif.). Each assay was carried out using 10 ng DNA in a 5-µL reaction consisting of TaqMan universal polymerase chain reaction master mix (Applied Biosystems, Foster City, Calif.), forward and reverse primers, and 6-carboxyfluorescein (FAM) and 4,7,2’-trichloro-7’-phenyl-6-carboxyfluorescein (VIC)-labeled probes designed by Applied Biosystems (ABI Assays-on-demand). Allelic discrimination was measured automatically using Sequence Detection Systems 2.1 software (automatic confidence level 95%). Approximately 8% of all genotypes were repeated in independent polymerase chain reactions to check for consistency and to ensure intraplate and interplate genotyping quality control. No genotyping discrepancies were detected between the repeated samples (Figure 1 ).
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The workflow of association analysis.
6
Genomic DNA Extraction and Genotyping
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Genomic DNA extraction from peripheral leucocytes were finished using a DNA isolation kit in accordance with the protocol (TIANGEN, Beijing, China) and quantified by a NanoDrop 2000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE). The final concentration of the samples ranged from 10 to 30 ng/mL. Probe and primer for rs35006907 genotyping were purchased from ThermoFisher (Assay ID: C____449430_10). The variant rs35006907 was genotyped using a TaqMan assay on the TaqMan 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) with the following condition: 10 min at 95 °C (enzyme activation) followed by 45 cycles at 95 °C for 15 s and 60 °C for 1 min (annealing/extension).
7
Genotyping of ARRB2 Variants
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We extracted genomic DNA from peripheral blood leukocytes using Tiangen commercially available kit (Tiangen, Beijing, China) according to the protocol. The DNA was stored at -80°C for further use. Referring to the Chinese data of the 1000 Genomes, we selected common genetic variants with minor allele frequency (MAF) >0.05 in the functional region of ARRB2 for further genotyping. The probe for genotyping came from ABI with the following assay ID: rs1045280 (C___8718195_20, 4351379). The common variant in ARRB2 was genotyped using the TaqMan assay on the TaqMan 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with the following conditions: 10 min at 95°C (enzyme activation) followed by 45 cycles at 95°C for 15 s and 60°C for 1 min (annealing/extension). An end point read was performed for allelic discrimination after amplification.
8
Genomic DNA Extraction and Genotyping
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We extracted genomic DNA from peripheral blood leukocytes using the Tiangen commercially available kit (Tiangen, Beijing, China). Detailed procedures of DNA extraction have been described previously [3] . Referring to the Chinese data of the 1000 Genomes, we selected common genetic variants with minor allele frequency >0.05 in the exon region of NEU1, NEU2, NEU3, and NEU4 for further genotyping. The probes for genotyping came from ABI with the following assay ID: rs2233384 (C__15962661_10, 4351379), rs2233385 (C__15962660_10, 4351379), rs2233394 (C_____42329_1_, 4351379), rs36111671 (C___2769365_10, 4351379), rs11545301 (C__57931136_10, 4351379), rs2293764 (C__16185067_10, 4351379), rs2293763 (C__16185022_10, 4351379), rs2293759 (C__11536383_1_, 4351379), rs544115 (C___1053082_10, 4351379). The genotyping procedure was completed according to the TaqMan assay on the TaqMan 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with the following conditions: 10 min at 95°C (enzyme activation) followed by 45 cycles at 95°C for 15 s and 60°C for 1 min (annealing/extension).
9
MTHFR Genotyping from Blood
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Genomic DNA was extracted from peripheral white blood cells using the phenol/ chloroform method. Genotyping of the MTHFR C677T and A1298C polymorphisms was performed using the Taqman 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). For quality control, genotyping was performed blinded to the case/control status of the subjects. In addition, a randomly chosen 10% of the samples were genotyped again by different individuals; the results of these assays were 100% concordant.
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