Cells were seeded in T75 culture flasks and treated as described above with 10 μM 5AZA. DNA was extracted and bisulfite converted, as described above. Pyrosequencing assays were designed using the PyroQ assay design software. A common tag was placed on either the forward or reverse primer (depending on the strand to be sequenced) and a common universal biotinylated primer was used for all reactions as previously described [27 (link)]. PCR was performed using a nested PCR for specific amplification and cycling conditions included denaturation at 95°C for 4 min, followed by 10 cycles of 94°C for 15 s, touchdown from 60°C to 50°C (−1°/cycle) for 15 s and 72°C for 20 s, followed by a further 30 cycles at 50°C annealing temperature. The second PCR used 2 μl of a 1:10 dilution of the first PCR as template and the same cycling conditions. All products were confirmed to be single bands by agarose gel electrophoresis. Methylation values were calculated as an average of all 10 CpG sites within each assay as determined by the Pyro Q-CpG Software (Biotage). Primers detailed in Supplementary Table S2.
Pyro q cpg software
Manufactured by Biotage
Sourced in Sweden
Pyro Q-CpG is a software designed for the analysis of DNA methylation data generated by the PyroMark Q24 system. It provides a platform for processing, visualizing, and interpreting DNA methylation patterns in a quantitative manner.
Automatically generated - may contain errors
Lab products found in correlation
Epitect bisulfite kit, by Qiagen (8 mentions)
Ez dna methylation kit, by Zymo Research (4 mentions)
Streptavidin sepharose bead, by GE Healthcare (3 mentions)
Psq 96ma pyrosequencer, by Biotage (3 mentions)
Pyromark q96 id pyrosequencing system, by Biotage (3 mentions)
Pyromark id system, by Biotage (2 mentions)
Kapa2g robust hot start taq dna polymerase, by EQT (2 mentions)
Pyromark cpg assay, by Qiagen (2 mentions)
Ez dna methylation gold kit, by Zymo Research (2 mentions)
Pyromark gold q96 reagent kit, by Qiagen (2 mentions)
Pyromark gold 96 reagent kit, by Qiagen (2 mentions)
Pyromark q96 system, by Biotage (2 mentions)
Psq96ma system, by Biotage (2 mentions)
Abi 9700 pcr system, by Thermo Fisher Scientific (1 mentions)
Pyromark q96 48 id, by Qiagen (1 mentions)
Psq 96ma, by Biotage (1 mentions)
Pyromark gold q96 reagent, by Qiagen (1 mentions)
Streptavidin coated sepharose beads, by GE Healthcare (1 mentions)
Psq 96ma instrument, by Biotage (1 mentions)
Pyromark assay design software 2, by Qiagen (1 mentions)
30 protocols using pyro q cpg software
1
Methylation Analysis by Pyrosequencing
2
Quantifying DNA Methylation by Pyrosequencing
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Sizeable, significant (P value <0.05 and |log2FC|>0.8, FC, fold change) DMRs were tested using pyrosequencing assay. Extracted DNA methylation was assessed using the Qiagen EpiTect Bisulfite kit (Qiagen, 59104). PCR amplification (ABI 9700 PCR System, Applied Biosystems) for 40 cycles was performed. Pyrosequencing was performed using PyroMark Q96/48 ID (Qiagen), and the final datasets were calculated using PyroQ CpG software (Biotage) according to the manufacturer's protocols [24 (link)].
3
Bisulfite Conversion and Pyrosequencing Analysis
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Bisulfite conversion of genomic DNA was performed with the EpiTect Bisulfite Kit (Qiagen). The targeted regions were amplified by PCR. The purified PCR products were sequenced with the PSQ 96MA (Biotage, Qiagen). The pyrosequencing results were analyzed using the Pyro Q‐CpG software (Biotage). The pyrosequencing service was provided by CPOS. The PCR and sequencing primers are listed in the Table S1 , Supporting Information.
4
Pyrosequencing of Imprinted Genes
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Bisulfite-treated DNA was PCR amplified using Snrpn- or Peg3-specific primers, one of which was biotinylated at its 5’ end (for sequences and annealing temperatures, see list of primers in Table 1 ). The PCR product was then bound to streptavidin-coated Sepharose beads (GE-Healthcare) through the biotin tag and denatured to generate single-stranded DNA to allow annealing of an internal sequencing primer (16 pmol of each per reaction) (see list of primers in Table 1 ). Pyrosequencing was performed in a PSQ™96MA instrument (Biotage) using 25 μl of amplified DNA product and PyroMark Gold Q96 reagents (Qiagen). Pyrosequencing data analysis was done using the Pyro Q-CpG software (Biotage).
5
Validation of Methylation Profiling by Pyrosequencing
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Array methylation results were validated by sodium bisulphite pyrosequencing. Briefly, pyrosequencing assays were designed to sequence the individual CpG dinucleotides within the DMROI (primers are listed in Supplementary Table 3 , available as Supplementary data at IJE online) using Pyromark Assay Design Software 2.0 (Qiagen, Hilden, Germany); assays were analysed (PSQ 96MA machine; Biotage, Uppsala, Sweden) and percentage methylation calculated using Pyro Q-CpG software (Biotage). The sequenced region for HES1 encompassed only 9 of 15 CpGs in the 920 bp BATMAN DMROI, due to sequence design constraints.
6
Epigenetic Predictor Development via Pyrosequencing
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Genomic DNA was isolated with the NucleoSpin Tissue kit (Macherey & Nagel, Düren, Germany) and bisulfite converted using the EZ DNA Methylation kit (Zymo Research, Irvine, CA, USA). Pyrosequencing was performed on a PyroMark ID System (Biotage, Uppsala, Sweden). Primers for pyrosequencing were designed with the PSQ assay design software (Biotage; Supplemental Table S3 ). DNAm levels were determined with the Pyro-Q-CpG Software (Biotage). To train the epigenetic predictor on pyrosequencing data we divided the pyrosequencing samples into a training and validation set (Supplemental Table S4 and Supplemental Data 2 ). The multivariable linear regression model based on DNAm levels (β-values) at the four CpGs in α = ALOX12 (cg03762994), β = DOK6 (cg25968937), γ = LTC4S (cg26683398) and δ = TNNI3K (cg05264232) was as follows: Finally, we used the R package caret52 (link) to perform 10-fold cross-validation on the training dataset.
7
Methylation Analysis of MCM2 and NUP37
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For validation of the methylation pattern of MCM2 enhancer region and NUP37 promoter region, genomic DNA from frozen HCC tissues and paired nontumor tissues samples (n = 6) were extracted using Blood and Tissue Kit (#69504, Qiagen). The concentration of genomic DNA from each sample was determined using NANO Quant infinite M200PRO (TECAN) and a total of 500 ng genomic DNA (A260/A280 ratios ranging from 1.8 to 2.0) from each sample was used for bisulfite DNA conversion using the EpiTect Bisulfite Kit (#59104, Qiagen) following the manufacturer’s handbook for user. After cleanup of bisulfite converted genomic DNA, we amplified the target sequence using ABI 9700 PCR System and performed quantitative pyrosequencing using the PyroMark Q96 ID (QIAGEN). The genomic DNA methylation percentage was calculated with using Pyro Q—CpG software (Biotage). The primer sequences used in PCR amplification and pyrosequencing, as well as the chromosomal location of each target sequence in this study were summarized in Additional file 1 : Table S2. Pyrosequencing was performed using the platforms from Geneland Biotech Co., Ltd (Shanghai, CN).
8
DNA Methylation Analysis by Pyrosequencing
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Bisulfite-modified DNAs were evaluated by pyrosequencing [35 (link)], using primers and conditions previously described [36 (link)]. DNA pyrosequencing provides the methylation status of single CpGs at the specific genomic region analyzed. The degree of methylation at each CpG position was determined from the ratio of C and T by the Pyro Q-CpG Software (Biotage AB). Pyrosequencing was performed on the sense and antisense strand of RASSF1A (nucleotides −163 to +262 in chromosome 3: 50353109–50353534, NC_0000003.10) and of RASSF1C (nucleotides −86 to +193 in chromosome 3: 50349706–50349985, NC_0000003.10).
9
DNA Methylation Analysis by Pyrosequencing
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Genomic DNA was extracted from VSM and liver cells, and mouse aortae and liver samples as described [28] . Briefly, DNA was treated with sodium metabisulphite using the EZ DNA methylation kit (ZymoResearch). The pyrosequencing reaction was carried out using primers listed in Table 2. Modified DNA was amplified using KAPA2G Robust Hot Start Taq DNA polymerase (Labtech). The amplified products were immobilised on streptavidin-sepharose beads (GE Healthcare UK Ltd.), washed, denatured and released into annealing buffer containing the sequencing primers (Table 2) (Biomers, Söflinger, Germany). Pyrosequencing was carried out using an SQA kit on a PSQ 96MA pyrosequencer (Biotage, Uppsala, Sweden). The percentage methylation at each CpG locus was determined using the Pyro Q CpG software (Biotage).
10
Quantifying DNA Methylation by Pyrosequencing
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Bisulfite-modified DNA was amplified by PCR in a 50 μl reaction volume, using the primers described in Additional file 4 and reagents supplied by Applied Biosystems. A 40 μl aliquot of each PCR product was used to perform the pyrosequencing reaction following the manufacturer’s protocol and as previously described [26 (link)]. Negative controls recommended by the manufacturer were used, as well as positive controls that included (i) DNA in vitro methylated using SssI CpG Methyltransferase (New England Biolabs, Hitchin, UK) following the manufacturer’s instructions, (ii) hypomethylated DNA, generated through PCR and a (iii) mixture of equal volumes of the above methylated and unmethylated controls. The methylation quantification was analysed by Pyro Q-CpG Software (Biotage, Uppsala, Sweden).
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