Quantitative estimation of BRAF V600E mutants was performed with the therascreen® BRAF Pyro Kit (Qiagen). Briefly, PCR amplification of region flanking amino acid 600 of BRAF was performed on a PTC-200 thermal cycler (MJ Research, Waltham, MA). 1 μl of each isolated DNA was analyzed per run. Pyrosequencing was performed on the PyroMark Q24 platform (Qiagen) using the PyroMark Gold Q24 reagents. Pyrograms were generated with the PyroMark Q24 software (v. 2.0.6.) and data were analyzed manually or with a plug-in tool provided by Qiagen. Sequences surrounding the site of interest served as normalization and reference peaks for quantification and quality control. Dispensation order was as follows: 5′-GCT ACT GTA GCT AGT ACG AAC TCA-3′. Two different “sequence to analyze” were used: 5′-YAY TGT AGC TAG ACS AAA AYC ACC -3′ or 5′-CHC TGT AGC TAG ACS AAA ATY ACC -3′ for manual analysis. Samples with 5% mutated alleles or more were scored as mutation positive.
Pyromark gold q24
Manufactured by Qiagen
Sourced in Germany
The PyroMark Gold Q24 is a laboratory instrument used for performing pyrosequencing analysis. It is designed to provide accurate and reliable DNA sequencing data. The core function of the PyroMark Gold Q24 is to facilitate the detection and analysis of DNA sequences through pyrosequencing technology.
Automatically generated - may contain errors
Lab products found in correlation
Pyromark q24, by Qiagen (7 mentions)
Ptc 200 thermal cycler, by Bio-Rad (1 mentions)
Pyromark q24 platform, by Qiagen (1 mentions)
Therascreen braf pyro kit, by Qiagen (1 mentions)
Pcr4 topo vector, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
Ez dna methylation kit, by Zymo Research (1 mentions)
Streptavidin mag sepharose beads, by GE Healthcare (1 mentions)
Epitect fast bisulphite conversion kit, by Qiagen (1 mentions)
8 protocols using pyromark gold q24
1
Quantitative BRAF V600E Mutation Detection
2
Methylation Assessment of PITX1 Gene
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We used different methods (pyrosequencing and bisulfite sequencing) to assess the PITX1 methylation status. Pyrosequencing was performed with PyroMark Gold Q24 reagents and a PyroMark Q24 pyrosequencing machine (QIAGEN, Hilden, Germany). The PCR primers used in this study were 5’-GGAATTTAGTTAGGTTGAGTGATAGTAG-3’ and 5’-AAAATCCTTAAAATCTTCCTTCTACAT-3’. The primer used for pyrosequencing was 5’-GTTGTTTTTTAATTAGTTTGGATT-3’. For bisulfite sequencing, the PCR primers used were 5’-GGGTTA GTTTAGGTAGTTTTTA-3’ and 5’-ACCATCATTTC TATCCCAATCC-3’ (231 bp). For sequencing of the bisulfite-PCR product, the DNA fragment was purified and cloned into a pCR4-TOPO vector (Invitrogen). Clones for subsequent sequencing were randomly selected.
3
Genotyping MSMB SNP rs10993994 by Pyrosequencing
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MSMB SNP rs10993994 was genotyped from the DNA samples using the pyrosequencing system. Pyrosequencing reactions were performed using the PyroMark Gold Q24 reagents (Qiagen, Germany) and the PyroMark Q24 instrument according to the manufacturer's recommendations. The DNA was extracted from patient's blood and amplified, and PCR products were immobilized on streptavidin-coated sepharose beads to obtain single-stranded DNA. After denaturation reactions, the biotinylated single-stranded PCR amplicons were isolated and allowed to hybridize with sequencing primers and finally incubated with DNA polymerase.
4
Bisulfite-Pyrosequencing for PPL Methylation
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To assess the PPL methylation status, bisulfite-pyrosequencing was performed with PyroMark Gold Q24 reagents and a PyroMark Q24 pyrosequencing machine (Qiagen, Hilden, Germany). The PCR primers used in this study were 5′-AGTTGATATTGGGAGTAGGTGTTA-3′ and 5′-CAAATTCCCTAAAAACCCCTCTTAA-3′. The primer used for pyrosequencing was 5′-GGGGTTTTAGAATATAGG-3′.
5
Quantitative DNA Methylation Analysis
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The pyrosequencing technique was used for quantitative analysis of DNA methylation. Cell samples for the pyrosequencing were prepared as follows: Neuro-2a-Agouti and Neuro-2a-Daz1 cells were seeded onto 10 cm plates (0.3 x 10 6 cells/cm) and incubated with 6 mL of cell maintenance medium at 37°C and 5% CO 2 overnight in an incubator. Next day, 1 mL of 5-azaC was added to each culture and the cells were further cultured for 3 days. After incubation, DNA and RNA were purified (All Prep DNA/RNA mini KIT; QIAGEN, Venlo, Netherlands), and 1 μg of the purified DNA was used for bisulfite treatment (Methyleasy Xceed kit, Human Genetic Signatures, Newtown, NSW, Australia). The DNA sequence to be analyzed was amplified from the bisulfite product using biotin-labeled one-sided PCR primers and made into a template. The template was used for pyrosequencing using PyroMark Q24 (QIAGEN). PyroMarkGoldQ24 (QIAGEN) was used as a reagent, and PyroMarkQ24 software (QIAGEN) was used to analyze the methylation data.
6
Methylation Analysis of LINE-1 Promoter
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The degree of methylation at CpG sites in the promoter-proximal region of LINE-1 was measured using a pyrosequencing-based assay, as described previously [26 (link)]. Bisulfite-treated DNA was prepared using the EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA) and amplified via PCR. The PCR products were sequenced using the Pyromark Q24 (Qiagen, Hilden, Germany) and PyroMark Gold-Q24 (Qiagen) reagent kits.
7
Pyrosequencing of DNA samples
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Pyrosequencing was performed on the Qseq instrument (Bio Molecular Systems) by use of the PyroMark Gold Q24 reagents (Qiagen) and Streptavidin Mag Sepharose beads (GE Healthcare) according to the manufacturer's protocol. The forward primers were used as pyrosequencing primers. Pyrosequencing data were analyzed by use of the Qseq analysis software version 2.0.11 (Bio Molecular Systems).
8
Methylation Analysis of MNG Samples
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DNA from 77 FTC, 25 FT-UMP and 42 FTA were used for bisulphite conversion with the EpiTect Fast Bisulphite Conversion Kit (Qiagen). The bisulphite-converted DNA was amplified using Qiagen PCR kit for 45 cycles of 30 s at 94°C, 30 s at 58°C and 30 s at 72°C. The targeted region is located between -578 and -541 bp, named Region A as previously described (Wang et al. 2016) . CpG methylation was quantified at eight sites within Region A using PyroMark Q24 with Pyromark Gold Q24 reagents, and the data were analysed using Pyro Mark Q24 software (Qiagen). A methylation index (MetI) was calculated for each individual sample. Twelve cases of MNGs were similarly analysed for comparison.
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