Genomic DNA was extracted from peripheral blood samples in all subjects. We used the same bisulfite pyrosequencing protocol as recently described in our epigenetic study of the IGFBP1 gene in T2D [19 (link)]. This protocol has been widely used for sensitive and quantitative universal pyrosequencing methylation analysis of CpG sites [29 (link)]. DNA methylation levels at six CpG sites at 5′-UTR of the IGFBP1 gene were determined by using PyroMark CpG assay (ENSG00000146678), a PyroMark PCR kit (Qiagen, Hilden, Germany) and PyroMark Q96 ID pyrosequencing system (Biotage, Uppsala, Sweden). To control the conversion efficiency of the bisulfite treatment and accuracy in methylation analyses, unmethylated bisulfite converted and unconverted DNA samples (Qiagen) were used.
Pyromark q96 id pyrosequencing system
Manufactured by Biotage
Sourced in Germany, Sweden
The PyroMark Q96 ID is a pyrosequencing system designed for DNA analysis. It utilizes a sequencing-by-synthesis approach to generate sequence information from DNA samples. The system is capable of performing automated, high-throughput DNA sequencing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Epitect bisulfite kit, by Qiagen (3 mentions)
Pyro q cpg software, by Biotage (3 mentions)
Pyromark gold q96 reagent kit, by Qiagen (2 mentions)
Pyromark gold 96 reagent kit, by Qiagen (2 mentions)
Pyromark q96 system, by Biotage (2 mentions)
Pyromark pcr kit, by Qiagen (1 mentions)
Pyromark cpg assay kit, by Qiagen (1 mentions)
Pyromark cpg assay, by Qiagen (1 mentions)
Gentra puregene tissue kit, by Qiagen (1 mentions)
Nanodrop 2000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
4 protocols using pyromark q96 id pyrosequencing system
1
Quantitative Methylation Analysis of IGFBP1
2
Epigenetic Analysis by Bisulfite Pyrosequencing
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An epigenetic analysis was performed by bisulfite pyrosequencing, which is a sensitive and accurate method for the detection of methylation. DNA was treated with sodium bisulfite using the EpiTect Bisulfite Kit (Qiagen), which was followed by the cleanup of bisulfite-converted DNA. PCR amplification was then performed with a PyroMark CpG Assay Kit (Qiagen) and a PyroMark Gold Q96 Reagent Kit (Qiagen) in a PyroMark Q96 system (Biotage AB, Uppsala Sweden). PyroMark PCR master mix includes HotStarTaq DNA polymerase and optimized PyroMark reaction buffer that contains 3 mM MgCl2 and dNTPs, 10x CoralLoad Concentrate, 5x Q-Solution, 25 mM MgCl2, and RNase-free water. The PCR amplicon covers the sequence of human chomosome 3q: NC_000003.12 (179148114.179240084). Nine CpG sites are located in the promoter region of the PIK3CA gene (-100 bps) as indicated with the bold letter ‘C ’ and recorded as CpG1-9 (Figure 6A ). Finally, the methylation levels of these CpG sites were detected with a PyroMark Gold 96 Reagent Kit (Qiagen) and a PyroMark Q96 ID pyrosequencing system (Biotage). The unmethylated and unconverted DNA samples (Qiagen) were used to control the conversion efficiency during the bisulfite treatment and the accuracy of the methylation analyses. PyroQ-CpG software (Biotage) was used for methylation data analysis.
3
Bisulfite Pyrosequencing for Epigenetic Analysis
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Epigenetic analysis was performed by bisulfite pyrosequencing, which is a sensitive and accurate protocol [34 (link),35 (link)]. DNA was treated with sodium bisulfite using EpiTect Bisulfite kit (Qiagen) and cleanup of bisulfite-converted DNA was done. PCR amplification was then carried out using PyroMark CpG assay (Qiagen) and PyroMark Gold Q96 Reagent kits (Qiagen) in a PyroMark Q96 system (Biotage AB, Uppsala Sweden). PyroMark PCR master mix includes HotStarTaq DNA polymerase and optimized PyroMark reaction buffer containing 3 mM MgCl2 and dNTPs, 10x CoralLoad Concentrate, 5x Q-Solution, 25 mM MgCl2, and RNase-free water. The PCR amplicon covers the sequence in human chromosome 8: 117962434 to 117962479 (version 37.56). There are four CpG sites in the SLC30A8 gene promoter region as indicated with the bold letter ‘C ’ and recorded as CpG1-4 (Figure 1 ). Finally, methylation levels of these CpG sites were detected by using the PyroMark Gold 96 Reagent kit (Qiagen) and a PyroMark Q96 ID pyrosequencing system (Biotage). The unmethylated and unconverted DNA samples (Qiagen) were used for control of conversion efficiency in bisulfite treatment and accuracy in methylation analyses. PyroQ-CpG software (Biotage) was used for methylation data analysis.
4
Genomic DNA Extraction and Methylation
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Extraction of genomic DNA from the tissue samples was performed by the Gentra Puregene tissue kit (Qiagen), which enables purification of high molecular weight DNA (100-200 kb) suitable for archiving. DNA concentrations were quantified using NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, USA). The scalable purification procedure gently removes contaminants and inhibitors and large-volume samples can be purified for genetic and epigenetic analyses.
DNA samples were first treated with sodium bisulfite using EpiTect bisulfite kit (Qiagen).
This kit gives complete conversion of unmethylated cytosine to uracil and subsequent purification in less than 6h. This highly sensitive technique utilizes an innovative protection against DNA degradation and ensures high conversion rates of over 99%.
The IGFBP2 gene (HGNC: 5471) is located in chromosome 2q35. For the analysis of DNA
Reagent kit (Qiagen) and PyroMark Q96 ID Pyrosequencing System (Biotage, Uppsala, Sweden). Pyrosequencing methylation analysis of CpG sites is a sensitive and accurate method [30, 31] . PyroQ-CpG software (Biotage) was used for methylation data analysis.
Unmethylated bisulfite converted and unconverted DNAs (Qiagen) were used for control of conversion efficiency of the bisulfite treatment and accuracy in methylation analyses.
DNA samples were first treated with sodium bisulfite using EpiTect bisulfite kit (Qiagen).
This kit gives complete conversion of unmethylated cytosine to uracil and subsequent purification in less than 6h. This highly sensitive technique utilizes an innovative protection against DNA degradation and ensures high conversion rates of over 99%.
The IGFBP2 gene (HGNC: 5471) is located in chromosome 2q35. For the analysis of DNA
Reagent kit (Qiagen) and PyroMark Q96 ID Pyrosequencing System (Biotage, Uppsala, Sweden). Pyrosequencing methylation analysis of CpG sites is a sensitive and accurate method [30, 31] . PyroQ-CpG software (Biotage) was used for methylation data analysis.
Unmethylated bisulfite converted and unconverted DNAs (Qiagen) were used for control of conversion efficiency of the bisulfite treatment and accuracy in methylation analyses.
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