Bisulphite pyro-sequencing was performed as described previously [11 (link)]. Briefly, 0.5 μg of genomic DNA was bisulfite-modified using the EZ DNA Methylation Kit (Zymo Research, Orange, CA), according to the manufacturer’s protocol. The bisulfite-modified DNA was subjected to PCR amplification using a tailed reverse primer in combination with a biotin-labeled universal primer. The PCR and sequencing primers were designed using PyroMark Assay Design 2.0 (Qiagen GmbH, Hilden, Germany). The ABCA1 TSS (−90 to +190) was PCR-amplified with specific primers (Additional file 1 : Table S1) in a 25-μl reaction containing 2× RBC SensiZyme Hotstart Taq premix (RBC Bioscience, Taiwan). Prior to pyro-sequencing, 1.5 μl of each PCR reaction was analyzed on 1% agarose gel. The pyro-sequencing was performed on the PyroMark Q24 instrument (Qiagen) using the Pyro Gold Reagents (Qiagen), according to the manufacturer’s protocol. The methylation level of 11 CpG sites, which are located −13 to +95 with respect to the TSS, was measured. The methylation percentage of each cytosine was determined using the fluorescence intensity of cytosines divided by the sum of the fluorescence intensity of cytosines and thymines at each CpG site. In vitro methylated DNA (Millipore) was included as positive control for pyro-sequencing.
Pyromark q24 instrument
Manufactured by Qiagen
Sourced in United States, Germany, Sweden
The PyroMark Q24 instrument is a pyrosequencing system designed for rapid DNA sequence analysis. It provides real-time and quantitative information about DNA sequences, enabling users to perform genetic analysis and evaluate DNA methylation patterns.
Automatically generated - may contain errors
Lab products found in correlation
Pyromark q24, by Qiagen (7 mentions)
Pyromark pcr kit, by Qiagen (7 mentions)
Ez dna methylation kit, by Zymo Research (6 mentions)
Hotstartaq dna polymerase, by Qiagen (3 mentions)
Pyromark assay design 2, by Qiagen (2 mentions)
In vitro methylated dna, by Merck Group (2 mentions)
Pyro gold reagents, by Qiagen (2 mentions)
Pyromaker assay design 2, by Qiagen (2 mentions)
Pyromark vacuum q24 workstation, by GE Healthcare (2 mentions)
Pyromark assay design software, by Qiagen (2 mentions)
Streptavidin sepharose high performance, by GE Healthcare (2 mentions)
Pyromark assay design software 2, by Qiagen (2 mentions)
Pcr buffer, by Qiagen (2 mentions)
Ez dna methylation direct kit, by Zymo Research (1 mentions)
Takara ex taq, by Takara Bio (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Pyromark q24 software version, by Qiagen (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
Therascreen kras pyro kit, by Qiagen (1 mentions)
Therascreen kras pyro kit handbook, by Qiagen (1 mentions)
27 protocols using pyromark q24 instrument
1
Bisulfite Pyrosequencing of ABCA1 Promoter
2
Methylation Analysis of ZNF671 Promoter
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After determination of the amount of gDNA, up to 500 ng of gDNA was subjected to bisulfite conversion using EZ DNA Methylation-Direct ™ Kit (ZYMO RESEARCH, USA). Pyrosequencing was performed to evaluate 2 CpG sites of ZNF671 promoter region (NC_000019.10: 57727411-57727425, ACGGTTTTATGGCGG) on bisulfite-treated DNA in 460 samples from training set. PCR amplification and pyrosequencing primers for target sites were designed using PyroMark Assay Design 2.0. Bisulfite-treated DNA was then amplified using TaKaRa Ex Taq (TaKaRa, Beijing, China). The primers were listed as follows: ZNF671-F:5′- GGTGGAGGTGTTGGGAAA -3′; ZNF671-R: 5′- CTAAAACACAAAAACTACAAACACTTTAC-3′; ZNF671-S: 5′- GTTTGATGTTTTGTAGGGA-3′.The reaction mixture including 13.5 μl of nuclease-free water, 2 μL of 10 × Ex Taq Buffer, 2 μL of dNTP, 0.4 μL former primer and reverse primer, 0.2 μL of Ex Taq HS and 1.5 uL of bisulfite-treated DNA. The PCR product act as a template in Pyrosequencing reactions, using the PyroMark Q24 instrument (Qiagen, MD) according to the manufacturer’s recommended protocol.43 (link) Raw data were analyzed using the Pyromark Q24 analysis software (Qiagen, MD). The average percentage of methylation across 2 CpG sites were obtained.
3
Pyrosequencing Analysis of LINE-1 Methylation
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Methylation of LINE-1 in MM1 cells was analyzed by pyrosequencing, as previously described [114 (link)]. Briefly, all required primers (i.e., forward, biotinylated-reverse, and sequencing primers) were designed using the PyroMaker Assay Design 2.0 software (Qiagen, Venlo, Netherlands), and sequences are provided in Supplementary Table S2 . Bisulfite-converted LINE-1 DNA fragments were PCR amplified using the PyroMark PCR kit (Qiagen, Venlo, Netherlands). Successful PCR amplification was assessed by TBE electrophoresis at 2% agarose gel as described above, after which the PyroMark Q24 Instrument (Qiagen, Venlo, Netherlands) was used to perform pyrosequencing. Biotinylated PCR products were immobilized on streptavidin-coated Sepharose beads (High Performance, GE Healthcare, Chicago, IL, USA), captured by the PyroMark vacuum Q24 workstation, washed, and denatured. Single-stranded PCR products were then released into a 24-well plate and annealed to the sequencing primer for 2 min at 80 °C. After completion of the pyrosequencing run, results were analyzed using the PyroMark Q24 software (Qiagen, Venlo, The Netherlands).
4
Pyrosequencing-based Targeted Genomic Analysis
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Forward, biotinylated-reverse and sequencing primers were designed using PyroMark Assay Design 2.0 software. Primer sequences are provided in Supplementary Table 4 . Targets of interest were PCR amplified using the PyroMark PCR kit (Qiagen) according to manufacturer's instructions. Successful PCR amplification was evaluated by TBE electrophoresis at 2% agarose gel and visualized by GelRed™ staining. Pyrosequencing was performed using the PyroMark Q24 instrument (Qiagen). Briefly, the biotinylated PCR products were immobilized on streptravidin-coated Sepharose beads (High Performance, GE Healthcare), captured by the PyroMark vacuum Q24 workstation, washed and denatured. Then single stranded PCR products were released into a 24-well plate and annealed to the corresponding sequencing primer for 2 min at 80°C. After the pyrosequencing run was finished, the results were analyzed using the PyroMark Q24 software (Qiagen).
5
Hippocampal DNA Methylation Profiling
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DNA was isolated from the hippocampus tissue using the classic phenol–chloroform method. Briefly, the tissue sample was incubated in lysis buffer (100 mM Tris–HCl pH 7.5, 20 mM EDTA, 165 mM NaCl, 1.0% SDS, 70 μg/ml proteinase K) at 55 °C overnight. The resulting lysate was treated with an equal volume of a phenol/chloroform/isoamyl alcohol mixture (Nacalai, Japan) and purified through ethanol precipitation. The concentration of the extracted DNA was measured using the NanoDrop ND-1000 Spectrophotometer.
Subsequently, 1 μg of DNA was subjected to bisulfite conversion using the EZ DNA Methylation-Gold Kit (Zymo Research, USA) following the manufacturer's protocols. The bisulfite-treated DNA was then used for PCR amplification using the PyroMark PCR kit (Qiagen, USA) according to the manufacturer's protocols. The pyrosequencing reaction was performed on a PyroMark Q24 instrument (Qiagen, USA). The resulting data were analyzed and quantified using the PyroMark Q24 software version 2.0.8 (Qiagen, USA). The PCR and pyrosequencing primers used were listed in TableS1 , designed with the PyroMark Assay Design software v2.0.2.5 (Qiagen, USA).
Subsequently, 1 μg of DNA was subjected to bisulfite conversion using the EZ DNA Methylation-Gold Kit (Zymo Research, USA) following the manufacturer's protocols. The bisulfite-treated DNA was then used for PCR amplification using the PyroMark PCR kit (Qiagen, USA) according to the manufacturer's protocols. The pyrosequencing reaction was performed on a PyroMark Q24 instrument (Qiagen, USA). The resulting data were analyzed and quantified using the PyroMark Q24 software version 2.0.8 (Qiagen, USA). The PCR and pyrosequencing primers used were listed in Table
6
BRAF V600E Mutation Detection
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BRAF V600E mutants were evaluated using pyrosequencing per the manufacturer’s instructions. PCR amplification was performed with a forward primer (5′- biotin- TTCTTCATGAAGACCTCACAGTAA-3′ ) and a reverse primer (5′- CCAGACAACTGTTCAAACTGATG-3′ ) on a C1000 thermal cycler (BIO-RAD, California, USA). The pyrosequencing reaction was performed with a sequencing primer (5′- GGACCCACTCCCATCGAGATTT-3′ ) on a Pyromark Q24 instrument (Qiagen). The produced pyrogram was analyzed with the PyroMark Q24 software (Qiagen) to distinguish mutant versus wild-type alleles by relative peak height.
7
Quantitative KRAS Mutation Analysis
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Quantitative analysis of KRAS mutations was performed by pyrosequencing in accordance with routine practice at the Department of Pathology at Fundación Jiménez Díaz University Hospital, using the CE-IVD marked Therascreen KRAS Pyro kit (Qiagen), according to the manufacturer’s protocols. Each PCR product was analyzed by pyrosequencing using the Therascreen KRAS Pyro reagents (Qiagen), Streptavidin Sepharose High Performance (GE Healthcare Bio-Science AB, Uppsala, Sweden), and a PyroMark Q24 instrument (Qiagen).
8
Pyrosequencing of KRAS and BRAF
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Mutations in KRAS (codons 12 and 13) and BRAF (V600E) genes were analyzed using a CE‐IVD marked PyroMark (Qiagen, Hilden, Germany) according to the manufacturer's protocols (Therascreen KRAS Pyro Kit Handbook, version 1, July 2011). The primers used in the present study were described previously.21 The cut‐off value for the mutation assay was 15% mutant alleles. Each PCR product was examined by pyrosequencing using PyroMark Gold Q96 reagents, Streptavidin Sepharose High Performance (GE Healthcare Bio‐Science AB, Uppsala, Sweden) and a PyroMark Q24 instrument (Qiagen) with PyroMark Q24 1.0.6.3 software.
9
Pyrosequencing Validation of Methylation Differences
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Biologically-relevant genes exhibiting a methylation difference of Δβ > 0.2 (hypermethylation) and Δβ < −0.2 (hypomethylation) between patient and control cerebellum were selected for pyrosequencing validation. Briefly, the required primers (i.e., forward, reverse, and sequencing primers) were designed using the PyroMaker Assay Design 2.0 software (Qiagen) according to the manufacturer’s instructions (Additional file 1 : Table S3). Bisulfite-converted DNA fragments were PCR amplified using the PyroMark PCR kit (Qiagen; 978703). Successful PCR amplification was assessed by tris-boric acid-EDTA (TBE) electrophoresis at 1.5% agarose gel, after which the PyroMark Q24 Instrument (Qiagen) was used to perform pyrosequencing. Biotinylated PCR products were immobilized on streptavidin-coated Sepharose beads (GE Healthcare; 17511301), captured by the PyroMark vacuum Q24 workstation, washed and denatured. Single-stranded PCR products were subsequently released into a 24-well plate and annealed to the sequencing primer for 5 min at 80 °C. After completion of the pyrosequencing run, results were analyzed using the PyroMark Q24 software (Qiagen). Graphical representation was performed with GraphPad Prism version 9.3.1.
10
Methylation Analysis of NCAM1 and NGF in Autism
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Utilizing the Immolase DNA Polymerase (Bioline) 50 ng of bisulfite, converted DNA were added to the PCR reaction, following the manufacturer's recommendations. Table 1 shows the forward and reverse primer sequences for each gene. The PCR product was subjected to agarose gel electrophoresis to test for the presence of a single PCR product. 15 μL of the verified biotinylated PCR product was used for each sequencing assay. DNA pyrosequencing was performed in a PyroMark Q24 instrument (Qiagen), following the manufacturer's guidelines, and subsequent methylation analysis was determined with the PyroMark Q24 Software (Qiagen). Sequencing primers for each gene are listed in Table 1 . In the light of the literature (30 (link)–33 (link)) and recent results from this group of children (27 (link)), two loci previously linked to ASD (NCAM1 and NGF) were selected for methylation analysis. Primers for NCAM1 were designed to amplify a 3′UTR region (UCSC region chr11:112,965,249–112,965,403) that included CpG positions for which changes in methylation levels have been previously described (34 (link)). Primers for NGF were obtained from Qiagen to amplify a region at the 5′UTR, near the non-transcribed first exon of the gene (UCSC region chr1:115,880,563–115,880,774) (Table 1 ).
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