DNA extraction was performed from 5 slides from FFPE tumoral fragments using the QIAamp DNA kit (Qiagen, Courtaboeuf, France). Only samples containing at least 60% of tumor cells were processed (neuropathologist confirmation). A total of 20 to 200 ng of DNA was treated with sodium bisulfite using the EpiJET Bisulfite Conversion kit and purified according to the specified protocol (Thermo Fischer Scientific, Inc.,Waltham, MA, USA). Bisulfit-modified DNA was amplified using ampliTaq Gold 360 Master mix (Applied Biosystems, Foster City, CA, USA) with a forward primer and a biotinylated reverse primer (Pyromark Q96 CpG MGMT, Qiagen, Courtaboeuf, France). Pyrosequencing was performed using PyroMark-Q48 advanced CpG Reagents and the sequencing primer (Pyromark Q96 CpG MGMT Qiagen) using the Pyromark Q48 Autoprep software on a PyroMark Q48 pyrosequencer (Qiagen, Courtaboeuf, France). Full details for the CpG location and the validation method can be found in the study by Quillien et al. [13 (link)].
Pyromark q48 advanced cpg reagent
Manufactured by Qiagen
Sourced in Germany
The PyroMark Q48 Advanced CpG Reagents are a set of reagents designed to be used with the PyroMark Q48 Autoprep instrument for the analysis of DNA methylation patterns. The reagents provide the necessary components for the Pyrosequencing process, which allows for quantitative analysis of DNA methylation at specific CpG sites.
Automatically generated - may contain errors
Lab products found in correlation
Pyromark q48 autoprep software, by Qiagen (6 mentions)
Pyromark pcr kit, by Qiagen (6 mentions)
Pyromark q48 autoprep system, by Qiagen (5 mentions)
Pyromark assay design software 2, by Qiagen (4 mentions)
Pyromark q48 autoprep, by Qiagen (4 mentions)
Truemethyl oxbs module, by Tecan (3 mentions)
Qubit ssdna assay, by Thermo Fisher Scientific (3 mentions)
Acetonitrile, by Thermo Fisher Scientific (3 mentions)
Pyromark q48 autoprep apparatus, by Qiagen (3 mentions)
Pyromark assay design software, by Qiagen (3 mentions)
Pyromark q48 magnetic beads, by Qiagen (3 mentions)
Pyromark assay design 2, by Qiagen (2 mentions)
Truemethyl magnetic beads, by Thermo Fisher Scientific (2 mentions)
Hotstartaq master mix, by Qiagen (2 mentions)
Pyromark q48 autoprep 2, by Qiagen (2 mentions)
Topred nucleic acid gel stain, by Romer Labs (2 mentions)
Amplitaq gold 360 master mix, by Thermo Fisher Scientific (1 mentions)
Pyromark q96 cpg mgmt, by Qiagen (1 mentions)
Qiaamp dna kit, by Qiagen (1 mentions)
Pyromark q48 pyrosequencer, by Qiagen (1 mentions)
20 protocols using pyromark q48 advanced cpg reagent
1
MGMT Promoter Methylation Assay
2
Bisulfite Pyrosequencing for DNA Methylation Analysis
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The bisulfite pyrosequencing was performed as explained earlier51 (link), with slight modifications (SI Text). The primers for the PyroMark assays were designed using the PyroMark Assay Design Software 2.0 (Qiagen, Hilden, Germany) (Supplementary Table S3 ). The annealing temperatures were 56 °C, 58 °C, and 56 °C for the CDKN2D, PSAT1, and RASSF1 assays, respectively. The product specificity was validated by gel electrophoresis and CpN methylation was quantified using the PyroMark Q48 Autoprep System (Qiagen) and the PyroMark Q48 Advanced CpG Reagents (Qiagen). Methylation values [%] were calculated with the PyroMark Q48 Autoprep 2.4.1 Software (Qiagen).
3
Methylation Analysis of Oligodendrocyte Precursors
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Genomic DNA was extracted from laser-captured OPCs, as well as transfected iPSC-OPCs, and bisulfite-converted, using the Zymo Research EZ DNA Methylation-Direct Kit (BaseClear Lab Products). PCR primers were designed using the PyroMark Assay Design 2.0 software (Qiagen, Supplementary Information S2). The assay for MBP was tested for its sensitivity using the EpiTect PCR Control DNA Set (Qiagen). Product amplification was performed using the following reaction mixture: 1 × buffer with 20 mM MgCl2 (Roche), 10 mM dNTP mix (Roche), 5 μM forward and reverse primers (Metabion AG), 1 U FastStart Taq DNA Polymerase (Roche), bisulfite-converted DNA, and nuclease-free water to a total volume of 25 μl. PCR cycling was performed as follows: initial denaturation for 5 min at 95 °C, 50 cycles of 30 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C; final extension for 7 min at 72 °C. PCR amplicons were sequenced using the Pyromark Q48 instrument (Qiagen) with the PyroMark Q48 Advanced CpG Reagents (Qiagen), according to the manufacturer’s protocol and quantified with the Pyromark Q48 Autoprep software.
4
Detecting Hot-Spot DNA Variants in LR-MDS
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To verify whether detected DNA-seq-var could be classified as hot-spots for LR-MDS/AML-MR, we performed SSeq and pyrosequencing genotyping within all 23 primary LR-MDS patients. The Sanger Sequencing was performed as described above. PyroMark Assay Design Software 2.0.1.15 (Qiagen) was used to design primers for pyrosequencing. The primer set was composed of primers for amplicon preparation (including one labelled with biotin at the 5’end) and sequencing primer (Table 1 ). The pyrosequencing tests were designed using the PyroMarkQ48 Autoprep 2.4.2 Software (Qiagen). Pyrosequencing was performed using PyroMarkQ48 Advanced CpG Reagents (Qiagen) and PyroMarkQ48 Autoprep (Qiagen), according to the manufacturer’s standard protocol, with quantified mean mutated AF determined. Sample positivity thresholds were calculated for each pyrosequencing test, based on healthy control results (5 PB). The allele frequency threshold was calculated as twice the standard deviation plus the highest AF value obtained for healthy controls. DNA sequence variants previously detected in BM were additionally genotyped in one matched PB and 3 SAL samples.
5
Pyrosequencing of CpG Sites
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Pyrosequencing was performed using PyroMark Q48 Advanced CpG Reagents (4 × 48) (Qiagen, Hilden, Germany) on a PyroMark Q48 autoprep instrument following the manufacturer’s instructions (Cat No./ID: 974,230). For each pyrosequencing run, 10 µL of biotinylated PCR product were bound to 3 µl PyroMark Q48 Magnetic Beads and were pipetted into the correct wells of the PyroMark Q48 Disc. 60 µL of sequencing primer were downloaded on the instrument in the mentioned cartridge. There are two other cartridges for nucleotides and other reagents such as denaturation solution, enzyme, substrate, and annealing buffer. All additional steps were carried out automatically by the instrument. Results were automatically analyzed using PyroMark Q48 Autoprep software.
6
Selective 5mC detection using oxBS conversion
7
Pyrosequencing of Amplified Products
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Amplified products underwent pyrosequencing, performed on PyroMark® Q48 Autoprep Pyrosequencing system (Qiagen), using pyrosequencing primer [5′-GGGGAGGGAGTTTTTTTAGG-3′] (Integrated Data Technologies), and PyroMark® Q48 Advanced CpG Reagents (Qiagen) and PyroMark® Q48 Magnetic Beads (Qiagen) based on manufacturer’s protocol.
8
PARP4 Gene Methylation Analysis
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The bisulfite pyrosequencing analysis was performed on two CpG sites within the promoter region of the PARP4 gene (cg18582260 and cg17117459), for methylation analyses. Each primer was designed using the PyroMark Assay Design SW 2.0 software (Qiagen). The primer sequences were: 5’-GGGGTTATAGGTGTGAGTTGTT-3’ (forward), and 5’-ATTAACCCAAAAAAAAACTAACATTTTACA-3’ (5’-biotinylated-reverse). Bisulfite-treated DNA was amplified using the PyroMArk PCR kit (Qiagen) in accordance with the instructions of the manufacturer. The biotinylated PCR product was bound to streptavidin Sepharose HP beads (Amersham Biosciences, Little Chalfont, UK) to prepare the ssDNA template for sequencing, following the sample preparation guide. The sequencing reaction was carried out on a PyroMark Q48 Autoprep system using PyroMark Q48 Advanced CpG Reagents (Qiagen) in accordance with the instructions of the manufacturer. The sequences we analyzed used 5’-GGGAGGTATGGAAAG-3’ as the sequencing primer.
9
Selective 5mC detection using oxBS conversion
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To selectively detect 5mC modification, genomic DNA was subjected to oxBS conversion (Booth et al., 2012 (link)) using TrueMethyl oxBS module (NuGEN Technologies) as per the manufacturer’s recommendations. In short, genomic DNA was affinity-purified using 80% acetonitrile (Fisher Scientific) and TrueMethyl magnetic beads to eliminate potential contaminating compounds. After the denaturation step, genomic DNA was oxidized to convert 5-hydroxymethylcytosine to 5-formylcytosine. Bisulfite treatment was then carried out to convert 5-formylcytosine to uracil, leaving 5-methylcytosine intact. Following desulfonation and purification, converted DNA was quantified using Qubit ssDNA assay (Invitrogen). PCR amplification of oxBS converted DNA was carried out with biotin-labeled primers. Primer design was carried out using PyroMark Assay Design software (version 2.0, QIAGEN). Pyrosequencing of biotinylated PCR products was performed using PyroMark Q48 Advanced CpG reagents (QIAGEN) on a PyroMark Q48 Autoprep apparatus (-QIAGEN) following the manufacturer’s protocol. 5mC levels at CpG sites were determined using PyroMark Q48 Autoprep software (version 2.4.2, QIAGEN) in CpG Assay mode. All samples were prepared, amplified and sequenced in triplicates. PCR and pyrosequencing primers are listed in Table S2 .
10
Quantifying SSTR2 Methylation Levels
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Three CpG sites at SSTR2 promoter were selected for the quantification of the extent of methylation. Bisulfite-modified DNA (100 ng) was PCR-amplified in a 20-μL reaction volume using MG 2× Taq Master Mix with Dye (MGmed, Seoul, Republic of Korea) to yield 119-bp product, using specific primers (Supplemental Table S1 ). PCR was performed with an initial melting step of 95 °C for 10 min; followed by 40 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s; with a final incubation at 72 °C for 7 min. Pyrosequencing was performed as described [19 (link)], using a sequencing primer (Supplemental Table S1 ) and PyroMark Q48 Advanced CpG Reagents (QIAGEN) with PyroMark Q48 Autoprep (QIAGEN).
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