Pyromark q24 advanced platform

Manufactured by Qiagen

Sourced in Germany

The PyroMark Q24 Advanced platform is a real-time pyrosequencing system designed for DNA analysis. It provides accurate and reliable sequence information for a wide range of applications, including gene expression analysis, epigenetics, and mutation detection.

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Lab products found in correlation

Pyromark assay design 2, by Qiagen (4 mentions) Pyromark q24 advanced cpg reagents, by Qiagen (3 mentions) Epitect bisulfite kit, by Qiagen (3 mentions) Pyromark q24 advanced software, by Qiagen (3 mentions) E z n a sq blood dna kit 2, by Omega Bio-Tek (2 mentions) Mastercycler, by Eppendorf (2 mentions) Pyromark reagents, by Qiagen (2 mentions) Pyromark assay design software 2, by Qiagen (2 mentions) Quantstudio 3, by Thermo Fisher Scientific (1 mentions) Taqpath proamp master mix, by Thermo Fisher Scientific (1 mentions) Pyromark q24 advanced reagents, by Qiagen (1 mentions) Hot start taq dna polymerase, by New England Biolabs (1 mentions) Ez dna methylation kit, by Zymo Research (1 mentions) Pyromark pcr kit, by Qiagen (1 mentions)

Close spelling variants of this product

PyroMark Q24 (386 citations) PyroMark Q24 platform (32 citations) PyroMark Q24 Advanced (24 citations) Pyromark platform (3 citations)

10 protocols using pyromark q24 advanced platform

Pyrosequencing Methylation Assays for DMRs

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Primers for pyrosequencing methylation assays of DMRs were designed using the PyroMark Assay Design 2.0 Software (Qiagen, NL). For repetitive regions, we could not anchor primers to non-repetitive flanking sequences because the closest non-repetitive regions were farther than two kb from the differentially methylated CG sites. Hence, both PCR primers were located within the repeat and, in principle, could amplify more than one copy of the transposon. The list of primers for pyrosequencing methylation assays is provided in Table S3.
250 to 1000 ng of DNA per sample were treated with sodium bisulfite using EpiTect Bisulfite Kit (Qiagen, NL). Pyrosequencing was carried out using the PyroMark Q24 Advanced platform and PyroMark Q24 Advanced CpG Reagents (Qiagen, NL). Results were analyzed using the PyroMark Q24 Advanced software (Qiagen, NL).

Batdorj E., AlOgayil N., Zhuang Q.K., Galvez J.H., Bauermeister K., Nagata K., Kimura T., Ward M.A., Taketo T., Bourque G, & Naumova A.K. (2022). Genetic variation in the Y chromosome and sex-biased DNA methylation in somatic cells in the mouse. Mammalian Genome, 34(1), 44-55.

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SNP rs4236 Expression Analysis

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We chose to analyse MGP transcript SNP rs4236 (T > C, OA risk allele = C, minor allele frequency = 0.37) as the difference in expression between its risk and non-risk alleles was the largest of those SNPs investigated in the original study [8 (link)]. The SNP was genotyped by pyrosequencing using the primers listed in Additional file 2: Table S2. The assay was designed using PyroMark assay design 2.0 (Qiagen), and the sequencing was performed using the PyroMark Q24 Advanced platform (Qiagen).

Shepherd C., Reese A.E., Reynard L.N, & Loughlin J. (2019). Expression analysis of the osteoarthritis genetic susceptibility mapping to the matrix Gla protein gene MGP. Arthritis Research & Therapy, 21, 149.

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Genotyping of SNPs by Pyrosequencing and RFLP

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Genotyping at rs10948172 was completed by pyrosequencing at proxy SNP rs529125 (r²=1.0). rs10948155 was genotyped directly by pyrosequencing. Genotype at rs62435998 was determined by a restriction fragment length polymorphism (RFLP) assay using the enzyme HaeIII, which cuts at the C allele of the SNP. Both pyrosequencing RFLP analysis were carried out in patient DNA samples following polymerase chain reaction (PCR) amplification of the region encompassing the SNP (Table S2). For pyrosequencing, PCR products were analysed using the PyroMark Q24 Advanced platform (Qiagen) along with the appropriate sequencing primer (Table S3) and the manufacturer’s recommended reagents.

Rice S.J., Aubourg G., Sorial A.K., Almarza D., Tselepi M., Deehan D.J., Reynard L.N, & Loughlin J. (2018). Identification of a novel, methylation-dependent, RUNX2 regulatory region associated with osteoarthritis risk. Human Molecular Genetics, 27(19), 3464-3474.

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Genotyping UGT1A1 Polymorphisms via PCR and Pyrosequencing

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Peripheral venous blood samples were collected into EDTA-anticoagulate tubes. Genomic DNA was extracted using a E.Z.N.A. ® SQ Blood DNA Kit II (Omega Bio-Tek, USA) according to the manufacturer's instructions and stored at -80 °C until use. Genotyping of UGT1A1 polymorphisms was carried out by polymerase chain reaction (PCR) and pyrosequencing as described previously [32] . Briefly, The DNA fragments flanking the UGT1A1*28 or *6 polymorphisms were amplified using Mastercycler (Eppendorf, Germany) in a final reaction volume of 50 µl, which contained 2 µl DNA, 5 µl PCR buffer, 1.5 µl dNTP, 0.5 ul DNA polymerase, 0.05 nM of each amplification primer (Additional file 1: Table S1), and 40 μl sterile double-distilled water. The thermal cycling of PCR was used as follows: degeneration at 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 58 °C or 66 °C for 30 s respectively, and 72 °C for 30 s; and a final extension 72 °C for 7 min. The PCR products were verified by agarose electrophoresis, followed by pyrosequencing on the PyroMark Q24 Advanced platform (Qiagen, Germany) using PyroMark Reagents (Qiagen, Germany) with the pyrosequencing primer (Additional file 1: Table S1). Genotyping results of each SNP were verified in 5% samples selected randomly using Sanger sequencing.

Díaz-Santa J., Rodríguez-Romanos R., Osca G., Pratcorona M., Garrido A., Coll R., Moret C., Escoda L., Tormo M., Heras I., Arnan M., Vives S., Salamero O., Lloveras N., Bargay J., Sampol A., Cruz D., Garcia A., Quiñones T., Esteve J., Sierra J, & Gallardo D. (2020). UGT1A1 genotype influences clinical outcome in patients with intermediate-risk acute myeloid leukemia treated with cytarabine-based chemotherapy. Leukemia, 34(11).

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SNP Genotyping by Multiple Techniques

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SNPs were genotyped by pyrosequencing (rs11783799 and rs11136345), by restriction fragment length polymorphism (RFLP) analysis (rs11780978 and rs7819099), or by a real‐time SNP genotyping assay (rs9100) (catalog no. 4351379; ThermoFisher Scientific). The rs11136336 SNP is in perfect linkage disequilibrium (r² = 1.0) with rs11783799; the rs11783799 assay was therefore used to genotype rs11136336. Pyrosequencing and RFLP polymerase chain reactions (PCRs) were performed using a G‐Storm GS4 Q4 Quad Block Thermal Cycler (Somerton Biotechnology) and the primers listed in Supplementary Table 2 (available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40849/abstract). Pyrosequencing assays were designed using PyroMark assay design software 2.0 (Qiagen), and the sequencing was performed using a PyroMark Q24 Advanced platform (Qiagen) with the recommended kit, following the instructions of the manufacturer. The RFLP‐digested fragments were separated by electrophoresis through a 3% agarose gel and visualized using ethidium bromide staining. The rs9100 real‐time genotyping assay was run on a QuantStudio 3 (Applied Biosystems), using TaqPath ProAmp MasterMix (ThermoFisher) following the instructions of the manufacturer.

Rice S.J., Tselepi M., Sorial A.K., Aubourg G., Shepherd C., Almarza D., Skelton A.J., Pangou I., Deehan D., Reynard L.N, & Loughlin J. (2019). Prioritization of PLEC and GRINA as Osteoarthritis Risk Genes Through the Identification and Characterization of Novel Methylation Quantitative Trait Loci. Arthritis & Rheumatology (Hoboken, N.j.), 71(8), 1285-1296.

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Pyrosequencing protocol for polymorphism analysis

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Amplicons were prepared for pyrosequencing on the PyroMark Q24 Advanced platform (QIAGEN) using PyroMark Q24 Advanced Reagents as per the manufacturer’s instructions. Codons for glycine 54 (Gly-54), leucine 98 (Leu-98), tyrosine 121 (Tyr-121), proline 216 (Pro-216), phenylalanine 219 (Phe-219), methionine 220 (Met-220), threonine 289 (Thr-289) and the region comprising promoter-associated TRs were analysed for polymorphisms (Figure 1) from January 2017. Codons glycine 138 (Gly-138), aspartic acid 427 (Asp-427), tyrosine 431 (Tyr-431), glycine 432 (Gly-432), glycine 434 (Gly-434) and glycine 448 (Gly-448) were screened from mid-September 2018. Individual assays were designed using the QIAGEN Assay Design software (QIAGEN) to span each polymorphism by de novo sequencing (SEQ) or allele quantification (AQ) depending on the multiplicity of the polymorphisms. SEQ assays were used for positions where multiple nucleotide polymorphisms were possible within one codon, whereas semi-quantitative AQ assays were designed for codons with multiple alleles at a single base-pair position (Figure 1 and Table S2). Both sense and anti-sense primers were used for pyrosequencing of 10 isolates during the validation phase, with results compared against Sanger sequencing of the same region.

Novak-Frazer L., Anees-Hill S.P., Hassan D., Masania R., Moore C.B., Richardson M.D., Denning D.W, & Rautemaa-Richardson R. (2020). Deciphering Aspergillus fumigatus cyp51A-mediated triazole resistance by pyrosequencing of respiratory specimens. Journal of Antimicrobial Chemotherapy, 75(12), 3501-3509.

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UGT1A1 Genotyping Protocol

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Peripheral venous blood samples were collected into EDTA-anticoagulate tubes. Genomic DNA was extracted using a E.Z.N.A.^® SQ Blood DNA Kit II (Omega Bio-Tek, USA) according to the manufacturer’s instructions and stored at − 80 °C until use. Genotyping of UGT1A1 polymorphisms was carried out by polymerase chain reaction (PCR) and pyrosequencing as described previously [32 (link)]. Briefly, The DNA fragments flanking the UGT1A1*28 or *6 polymorphisms were amplified using Mastercycler (Eppendorf, Germany) in a final reaction volume of 50 µl, which contained 2 µl DNA, 5 µl PCR buffer, 1.5 µl dNTP, 0.5 ul DNA polymerase, 0.05 nM of each amplification primer (Additional file 1: Table S1), and 40 μl sterile double-distilled water. The thermal cycling of PCR was used as follows: degeneration at 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 58 °C or 66 °C for 30 s respectively, and 72 °C for 30 s; and a final extension 72 °C for 7 min. The PCR products were verified by agarose electrophoresis, followed by pyrosequencing on the PyroMark Q24 Advanced platform (Qiagen, Germany) using PyroMark Reagents (Qiagen, Germany) with the pyrosequencing primer (Additional file 1: Table S1). Genotyping results of each SNP were verified in 5% samples selected randomly using Sanger sequencing.

Chen P., Zhu K.W., Zhang D.Y., Yan H., Liu H., Liu Y.L., Cao S., Zhou G., Zeng H., Chen S.P., Zhao X.L., Yang J, & Chen X.P. (2018). Influence of UGT1A1 polymorphisms on the outcome of acute myeloid leukemia patients treated with cytarabine-base regimens. Journal of Translational Medicine, 16, 197.

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DNA Methylation Analysis via Bisulfite Pyrosequencing

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DNA was treated with sodium bisulfite using EpiTect Bisulfite Kit (Qiagen, NL). Primers for differentially methylated regions (DMRs) (50–70 bp) were designed using the PyroMark Assay Design 2.0 Software (Qiagen, NL). PCR was conducted using the Hot Start Taq DNA polymerase (New England Biolabs, MA, USA). The list of primers is provided in Supplementary Table S3. Pyrosequencing was carried out using the PyroMark Q24 Advanced platform and PyroMark Q24 Advanced CpG Reagents (Qiagen, NL). Results were analyzed using the PyroMark Q24 Advanced software (Qiagen, NL).

AlOgayil N., Bauermeister K., Galvez J.H., Venkatesh V.S., Zhuang Q.K., Chang M.L., Davey R.A., Zajac J.D., Ida K., Kamiya A., Taketo T., Bourque G, & Naumova A.K. (2021). Distinct roles of androgen receptor, estrogen receptor alpha, and BCL6 in the establishment of sex-biased DNA methylation in mouse liver. Scientific Reports, 11, 13766.

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Sex-specific DNA Methylation Analysis

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One thousand nanograms of DNA per sample was treated with sodium bisulfite using EpiTect Bisulfite Kit (Qiagen, NL, USA). Primers for sex-associated differentially methylated regions (sDMRs) (50–70 bp) were designed using the PyroMark Assay Design 2.0 Software (Qiagen, NL, USA). The list of primers is provided in Table S2. Pyrosequencing was carried out using the PyroMark Q24 Advanced platform and PyroMark Q24 Advanced CpG Reagents (Qiagen, NL, USA). The results were analyzed using the PyroMark Q24 Advanced software (Qiagen, NL, USA).

Zhuang Q.K., Galvez J.H., Xiao Q., AlOgayil N., Hyacinthe J., Taketo T., Bourque G, & Naumova A.K. (2020). Sex Chromosomes and Sex Phenotype Contribute to Biased DNA Methylation in Mouse Liver. Cells, 9(6), 1436.

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Bisulfite Conversion and Pyrosequencing of MSCs

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DNA was isolated from Day0 MSCs and Day14 cartilagenous discs as above then 500 ng bisulphite converted with the EZ DNA Methylation Kit (Zymo Research, California, USA) following the manufacturer’s instructions. Pyrosequencing PCR was performed using the PyroMark PCR Kit (Qiagen) in a 20 µl reaction volume in duplicate with 1 µl bisulphite-converted DNA following the manufacturer instructions. Pyrosequencing assays were designed using PyroMark assay design software 2.0 (Qiagen), and the sequencing was performed using a PyroMark Q24 Advanced platform (Qiagen) with the recommended kit, following the instructions of the manufacturer and as previously detailed^{60 (link)}. Sequences of forward and biotinylated-reverse PCR primers and sequencing primers are listed in Supplementary Table 5.

Barter M.J., Bui C., Cheung K., Falk J., Gómez R., Skelton A.J., Elliott H.R., Reynard L.N, & Young D.A. (2020). DNA hypomethylation during MSC chondrogenesis occurs predominantly at enhancer regions. Scientific Reports, 10, 1169.

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