Genotyping was performed at the Children’s Hospital Boston using a custom Sequenom iPLEX assay in conjunction with the Mass ARRAY platform (Sequenom Inc., La Jolla, CA, USA). One panel of SNP markers was designed using SEQUENOM ASSAY DESIGN 3.2 software (Sequenom Inc., La Jolla, CA, USA).
Sequenom assay design 3
Manufactured by Labcorp
Sourced in United States
The Sequenom Assay Design 3.1 is a software tool used for the design of genetic assays. It facilitates the creation of customized molecular assays by providing a platform for the automated design of primers and probes. The core function of this software is to enable the efficient development of targeted genetic analysis tools.
Automatically generated - may contain errors
Lab products found in correlation
Massarray platform, by Labcorp (3 mentions)
Massarray system, by Labcorp (3 mentions)
Tianamp blood dna kit, by Tiangen Biotech (3 mentions)
Iplex assay, by Labcorp (2 mentions)
Massarray iplex gold kit, by Labcorp (2 mentions)
Massarray typer 4, by Labcorp (1 mentions)
Guanidine hydrochloride, by Merck Group (1 mentions)
Massarray typer version 4, by Labcorp (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Ptc 100pcr, by Bio-Rad (1 mentions)
Kapa taq hotstart dna polymerase, by Roche (1 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions)
Massarray typer software version 3, by Labcorp (1 mentions)
Ezna blood dna midi kit, by Omega Bio-Tek (1 mentions)
12 protocols using sequenom assay design 3
1
Genotyping with Sequenom iPLEX Assay
2
Genotyping of DNA Samples via Sequenom
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Individual genotyping for selected markers in individual DNA samples was performed using a custom Sequenom iPLEX assay in conjunction with the Mass ARRAY platform (Sequenom Inc., La Jolla, CA, USA). Panels of SNP markers were designed using Sequenom Assay Design 3.2 software (Sequenom Inc.), in a similar fashion to the previously described methodology from our laboratory (9, 16, 17) .
3
Multiplex SNP Genotyping Using Sequenom
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sequence-tagged sites containing SNP information developed46 (link) were used as queries in blastn searches against Glyma1.01 with default parameters. Multiplex assays for 1,000 randomly selected SNPs distributed throughout the genome (Supplementary Table S3 ) were designed to amplify low-copy sequences in Sequenom Assay Design 3.1 software (Sequenom). The Sequenom MassARRAY system47 (link) was used for SNP genotyping. Multiplex PCR followed by template-directed single base extension at each SNP site was conducted with a MassARRAY iPLEX Gold kit (Sequenom) following the manufacturer‘s protocol. The genotypes were determined in MassARRAY Typer 4.0 software (Sequenom).
4
Genomic DNA Extraction and SNP Genotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
As described previously (Khosla et al., 1999 (link)), total genomic DNA was extracted from young fresh leaves (3 g) taken from the parental cultivars, RILs, first F1, first F2, second F1, and second F2 plants using guanidine hydrochloride (Sigma-Aldrich) and proteinase K (QIAGEN). Multiplex assays for 513 SNPs, distributed throughout the genome, were designed (Supplementary Table 2 ) using Sequenom Assay Design 3.1 (Sequenom). Genotyping was conducted using the Sequenom MassARRAY system (Oeth et al., 2009 (link)). Multiplex PCR, followed by template-directed single-base extension at each SNP, was conducted using the MassARRAY iPLEX Gold kit (Sequenom), following the instructions of the manufacturer. Genotypes were determined using MassARRAY Typer version 4.0 (Sequenom).
5
RANKL/RANK/OPG Pathway Genotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primers for polymerase chain reaction and sequencing were designed by Sequenom Assay Design 3.1 software (Sequenom, San Diego, CA, USA) according to the manufacturer's instructions, shown in Table 2 . The quality inspection of the sequencing primer was completed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). Mass ARRAY® platform (Sequenom Analyzer 4, Inc., San Diego, CA, USA) was used to analyze the 10SNPs gene polymorphisms of 5 genes in the RANKL/RANK/OPG signaling pathway in 200 ONFH patients and 177 healthy controls. The genotyping success rates for the 10 tag SNPs were >95%, respectively.
6
Genotyping of IL10 Polymorphisms in Liver Cirrhosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately 5-mL peripheral blood samples were drawn from the liver cirrhosis patients and the controls, and the blood samples were stored at -80°C until required. Genomic DNA was extracted from the peripheral blood using a TIANamp Blood DNA Kit (Tiangen Biotech, Beijing, China).
After extraction, genotyping of IL10 rs1800896 and rs1800872 polymorphisms was carried out by polymerase chain reaction coupled with restriction fragment length polymorphism. Primer sequences for IL10 rs1800896 and rs1800872 were designed using the Sequenom Assay Design 3.1 software (Sequenom, San Diego, CA, USA). The forward and reverse primers for IL10 rs1800896 were 5'-AGGATGTGTTCCAGGCTCCT-3' and 5'-CCCTTGTACAGGTGATGTAACA-3', respectively; the forward and reverse primers for rs1800872 were 5'-GGTGAGCACTACCTGACTA GC-3' and 5'-CCTAGGTCACAGTGACGTGG-3', respectively. The restriction enzymes for IL10 rs1800896 and rs1800872 were BseRI and RsaI, respectively. The digested fragments of the IL10 rs1800896 G allele were 33 and 106 bp, and those of the A allele were 33, 106, and 139 bp. The digested fragments of the rs1800872 allele were 176 and 236 bp, and those of the C allele were 176, 236, and 412 bp. Additionally, approximately 10% of the samples were randomly selected and retested, and the results were 100% concordant.
After extraction, genotyping of IL10 rs1800896 and rs1800872 polymorphisms was carried out by polymerase chain reaction coupled with restriction fragment length polymorphism. Primer sequences for IL10 rs1800896 and rs1800872 were designed using the Sequenom Assay Design 3.1 software (Sequenom, San Diego, CA, USA). The forward and reverse primers for IL10 rs1800896 were 5'-AGGATGTGTTCCAGGCTCCT-3' and 5'-CCCTTGTACAGGTGATGTAACA-3', respectively; the forward and reverse primers for rs1800872 were 5'-GGTGAGCACTACCTGACTA GC-3' and 5'-CCTAGGTCACAGTGACGTGG-3', respectively. The restriction enzymes for IL10 rs1800896 and rs1800872 were BseRI and RsaI, respectively. The digested fragments of the IL10 rs1800896 G allele were 33 and 106 bp, and those of the A allele were 33, 106, and 139 bp. The digested fragments of the rs1800872 allele were 176 and 236 bp, and those of the C allele were 176, 236, and 412 bp. Additionally, approximately 10% of the samples were randomly selected and retested, and the results were 100% concordant.
7
Genotyping of ABCB1 Polymorphisms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each patient was asked to provide a sample of 5 mL venous blood, which was stored at -20°C. Genomic DNA was extracted from venous blood using the TIANamp blood DNA kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. Genotyping of ABCB1 C3435T and G2677T/A was conducted using polymerase chain reaction (PCR)-restriction fragment length polymorphism. Primers for ABCB1 C3435T and G2677T/A were designed using the Sequenom Assay Design 3.1 software (Sequenom, San Diego, CA, USA). The PCR included the following steps: 94°C for 2 min, 35 cycles at 94°C for 30 s, with the annealing temperature reduced to 64°C for 30 s, and 72°C for 1 min. For ABCB1 C3435T, the PCR product was 244 bp and digested with MboI. For G2677T/A, the PCR product was 224 bp and digested with BanI. Finally, DNA sequencing was performed to validate the genotyping results.
8
Genomic DNA Extraction from FFPE Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tissues from patients with AGC were obtained via biopsy or surgery, fixed with 10% neutral buffer formalin for 24 h at room temperature, immersed in 60°C paraffin, embedded in a paraffin block and stored at 4°C. Genomic DNA was extracted from paraffin-embedded tissues of patients with AGC using the QIAamp DNA FFPE Tissue kit (Qiagen GmbH) according to the manufacturer's instructions. The polymerase chain reaction (PCR) primers for SNPs were designed using Sequenom Assay Design 3.1 software (Sequenom) and are listed in Table SII . A thermocycler (PTC-100PCR; MJ Research) and KAPA Taq HotStart DNA polymerase (Kapa Biosystems; Roche Diagnostics) were used for PCR amplification, the thermal cycling program employed was as follows: 94°C for 5 min, followed by 35 cycles of 30 sec at 94°C, then 30 sec of annealing at 60°C, 30 sec of extension at 72°C, and a final elongation step at 72°C for 10 min. The PCR products were sequenced using a 3730XL DNA Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.).
9
Multiplexed SNP Genotyping Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A multiplexed SNP single base extension (SBE) assay was designed by using Sequenom Assay Design 3.1 software (Sequenom, San Diego, CA) according to the instructions. Genotyping was performed by using a 384-well plate format on the Sequenom MassARRAY platform (Bioyong Technologies Inc., Beijing, China) during the year 2012. The raw data files generated by Mass Array Sequenom were analyzed for the intensity peaks of calibrant to ascertain the quality of the data as previous described [19 (link)]. An overall call rate of >95% was maintained. For every 96 samples (a quadrant of the Sequenom chip), four samples were duplicated and the call rates were checked for concordance. The calls in the negative control (no DNA) were also monitored in all the runs. Reproducibility was 100% in the present study.
10
Genotyping of SFRP4 and GSK3β Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primers for polymerase chain reaction and sequencing were designed by Sequenom Assay Design 3.1 software (Sequenom, San Diego, CA, USA) following the manufacturer's instructions, shown in Table 6 .The quality inspection of the sequencing primer was completed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). Mass ARRAY®platform (Sequenom Analyzer 4, Inc., San Diego, CA, USA) was used to analyse the 4 variants polymorphisms in the SFRP4 and GSK3β genes. The genotyping success rates for the 4SNPs were > 95%, respectively. The linkage disequilibrium analysis and Call Cluster Plot of the 4SNPs are presented in Figure 1 and Figure 2 , respectively.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!