Pyrosequencing of the KRAS codon 12/13 region was performed using the Therascreen KRAS Pyro Kit (Qiagen) as recommended by the manufacturer. 2 ng of DNA were used per analysis. PCR amplification of the target region was performed on a T-100 thermocycler (Biorad). For the pyrosequencing reaction on the PyroMark Q24 platform (Qiagen), amplicons were immobilized to the wells of a PyroMark Q24 plate using streptavidin high performance beads (GE Healthcare). Pyrosequencing results were analysed using the PyroMark Q24 software version 2.0 with the Therascreen KRAS Pyro-plugin report, which already incorporated the thresholds for mutation calls (detection limit for the mutation (LOD) + 3 %).
Pyromark q24 platform
Manufactured by Qiagen
Sourced in Germany, United Kingdom
The PyroMark Q24 platform is a pyrosequencing system designed for DNA sequencing and analysis. It is capable of generating real-time sequence data and quantitative information from short DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Pyromark q24, by Qiagen (9 mentions)
Pyromark gold q24 reagent, by Qiagen (5 mentions)
Therascreen braf pyro kit, by Qiagen (2 mentions)
Epitek bisulfite kit, by Qiagen (2 mentions)
4 topo vector, by Thermo Fisher Scientific (2 mentions)
Ez dna methylation gold kit, by Zymo Research (2 mentions)
Ez dna methylation kit, by Zymo Research (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Therascreen kras pyro kit, by Qiagen (1 mentions)
T100 thermocycler, by Bio-Rad (1 mentions)
M sssi, by New England Biolabs (1 mentions)
Pyromark gold q24, by Qiagen (1 mentions)
Ptc 200 thermal cycler, by Bio-Rad (1 mentions)
Qiaamp dna extraction kit, by Qiagen (1 mentions)
Pyromark assay design software v2, by Qiagen (1 mentions)
Pyromark assay design, by Qiagen (1 mentions)
Cpgenome universal methylated dna, by Merck Group (1 mentions)
Genomeplex, by Merck Group (1 mentions)
Pyromark assay design software 2, by Qiagen (1 mentions)
Pyromark assay design software, by Qiagen (1 mentions)
32 protocols using pyromark q24 platform
1
KRAS Codon 12/13 Pyrosequencing
2
Quantifying mtDNA Mutation Heteroplasmy
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The m.7486G>A mt-tRNA mutation load was assessed by pyrosequencing technology, in all available tissues and in individual COX-positive and COX-deficient muscle fibres. The PyroMark Assay Design Software v.2.0. (Qiagen) was used to design locus-specific PCR and sequencing primers for the m.7486G>A variant (biotinylated forward primer: m.7466–7485; reverse primer: m.7583–7600; sequence primer: m.7488–7502) and pyrosequencing was performed on the Pyromark Q24 platform, according to the manufacturer's protocol. Pyromark Q24 software was used to quantify the m.7486G>A heteroplasmy levels by directly comparing the peak heights of both wild-type and mutant nucleotides at this position [42] (link).
The multiplex MTND1/MTND4 real-time PCR assay was performed using DNA from individual COX-deficient and COX-positive isolated muscle fibres and muscle homogenate and the mtDNA deletion level was calculated from the proportion of wild-type (MTND4) to total (MTND1) copy number by the established ΔΔCt method [43] (link). PCR amplification was completed in a 25 µl reaction in triplicate for each sample, with each plate containing a serial dilution of p7D1 plasmid for standard curve generation, as reported previously [44] (link).
The multiplex MTND1/MTND4 real-time PCR assay was performed using DNA from individual COX-deficient and COX-positive isolated muscle fibres and muscle homogenate and the mtDNA deletion level was calculated from the proportion of wild-type (MTND4) to total (MTND1) copy number by the established ΔΔCt method [43] (link). PCR amplification was completed in a 25 µl reaction in triplicate for each sample, with each plate containing a serial dilution of p7D1 plasmid for standard curve generation, as reported previously [44] (link).
3
Quantification of Global DNA Methylation
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LUMA was used to measure global levels of DNA methylation by restriction digestion using the methylation sensitive HpaII/MspI isoschizomers and quantification of the resulting overhangs by pyrosequencing.36 (link), 37 (link) Individual samples were digested with both HpaII+EcoRI and MspI+EcoRI, (Fermentas) as separate reactions, where the EcoRI functions as an internal control. In total, 500 ng of genomic DNA was used for the individual restrictions, incubated for 4 h at 37 °C. Lambda DNA and in vitro CpG methyltransferase (M.SssI; New England Biolabs) treated Lambda DNA were used as controls. Samples were run on a Pyromark Q24 platform (Qiagen, KJ Venlo, The Netherlands) in duplicate. Peak heights were normalized to EcoRI to calculate global methylation levels as previously described.36 (link), 37 (link)
4
Quantitative BRAF V600E Mutation Detection
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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.
5
Mitochondrial DNA Sequencing and Analysis
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Total genomic DNA was isolated from blood, urine sediments and renal samples using the QIAamp DNA extraction kit (QIAGEN). The 16.5 kb mtDNA was amplified using 15 polymerase chain reaction (PCR) primer pairs and then sequenced. The sequencing data were compared against the revised Cambridge Reference Sequence (rCRS) of human mtDNA (GenBank NC_012920.1). For detection of mtDNA deletion, long range PCR was amplified using two primer pairs and the amplified DNA fragments were separated by electrophoresis on a 0.8% agarose gel. Real-time quantitative PCR was used to determine mtDNA copy number, relative to the nuclear 18S rRNA gene, using two primers specific of the mitochondrial COX1 locus and two primers specific of the nuclear 18SrRNA locus. The Pyromark Q24 platform (Qiagen) was used to assess the levels of heteroplasmy of the G8969>A mutation. For this, a fragment around position 8969 was PCR amplified with two primers firstly; then pyrosequencing of the amplificate was performed using the sequencing primer. The presence of the G8969>A mutation was also analyzed in 100 controls. All the primers used in this study were listed in Supplementary Table S1 .
6
Methylation Analysis of K+ Channel Genes
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To investigate the methylation pattern of CpG sites near the transcriptional start site (TSS) of four K+ channel genes, methylation data obtained from the L5 and L6 DRGs of control and SNL rats 3 weeks after surgery were compared. Genomic DNA obtained from pools of DRGs was treated with bisulfite using an Epitek bisulfite kit (Qiagen). This process converts the unmethylated cytosine to uracil but leaves the methylated cytosine unchanged. Using specific primers, the regions of interest close to the TSS of Kcna4, Kcnd2, Kcnq2, and Kcnma1 were amplified by PCR. The amplified DNA was cloned into 4-TOPO vector (Invitrogen), and the DNA obtained from individual clones after bacteria transformation was sequenced (Beckman Coulter Genomics). Each sequence, corresponding to a different allele, was analyzed for the presence of methylated cytosines using the QUMA web-based program (http://quma.cdb.riken.jp/ ). Primer sequences for bisulfate sequence are listed in Supplementary Table 4 . Bisulfite-treated DNA obtained from the same samples was also analyzed by pyrosequencing technology using the PyroMarkQ24 platform (Qiagen).
7
Mutational Analysis of BRAF and NRAS
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Pyrosequencing of mutation hotspots in BRAF and NRAS was performed on a PyroMark Q24 platform (Qiagen), using PyroMark Gold Q24 Reagents (Qiagen). The primer sequences are listed in Additional file 1 : Table S1.
8
Quantitative Pyrosequencing of mtDNA Mutations
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The mtDNA mutation load for specific alleles was assessed using quantitative
pyrosequencing. The Pyromark Assay Design Software v2.0 (Qiagen, Crawley, West
Sussex, UK) was used to design locus-specific PCR and pyrosequencing primers
(Supplementary Table S2 online) for each variant, and pyrosequencing was
performed on the Pyromark Q24 platform according to the manufacturer's
protocol. Quantification of the heteroplasmy level of each variant was achieved
using Pyromark Q24 software to directly compare the relevant peak heights of the
wild-type and the mutant nucleotides at the relevant position, as described
previously.27 (link)
pyrosequencing. The Pyromark Assay Design Software v2.0 (Qiagen, Crawley, West
Sussex, UK) was used to design locus-specific PCR and pyrosequencing primers
(
performed on the Pyromark Q24 platform according to the manufacturer's
protocol. Quantification of the heteroplasmy level of each variant was achieved
using Pyromark Q24 software to directly compare the relevant peak heights of the
wild-type and the mutant nucleotides at the relevant position, as described
previously.27 (link)
9
Methylation Analysis of K+ Channel Genes
10
Replication of Cartilage Methylation Profiles
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CpGs with nominal P < 0.05 in the mQTL discovery were replicated in an independent cohort of cartilage arthroplasty samples and in fetal cartilage samples. DNAs were genotyped at rs1046934 by pyrosequencing. For methylation quantification, 500 ng of DNA was bisulfite converted using EZ DNA methylation kits (Zymo Research). The CpG regions were PCR amplified in bisulfite–converted DNA with methylation levels quantified using the PyroMark Q24 Platform (Qiagen). Duplicate measures were performed and excluded if the difference was >5%. Oligonucleotide sequences, which were generated by PyroMark Assay Design (Qiagen), were obtained from IDT (Supplementary Table 4 , available at https://onlinelibrary.wiley.com/doi/10.1002/art.42427 ).
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