For quantitative methylation-specific PCR (qMSP) analysis, DNA (2 μg) was modified by bisulfite with a DNA methylation kit (Zymo Research, Orange, CA, USA), which converts nonmethylated cytosine to uracil and protects methylated cytosine. The methylation of gene promoter sites was assessed using qMSP primer pairs located close to the putative transcription start site in the 5′ CpG island. Bisulfite-treated DNA as the template and JumpStart REDTaq DNA polymerase (Sigma-Aldrich Co.) were used for amplification. For bisulfite sequencing, the PCR products were purified with a gel extraction kit (Qiagen GmbH, Hilden, Germany) and cloned using the TOPO TA vector system (Invitrogen, Carlsbad, CA, USA). Each clone was isolated and purified using a NucleoSpin plasmid isolation kit (Macherey-Nagel, Düren, Germany). Randomly selected positive clones (10–15 from each sample) were sequenced using the M13F primer, and the methylation status of each CpG dinucleotide was analyzed. For the quantification of p16INK4A methylation, qMSP amplification was performed on bisulfite-treated samples and normalized based on Alu element amplification. qPCR was performed using a CFX96TM real-time system (BioRad, Hercules, CA, USA). MSP and bisulfite sequencing primers were from published reports24 (link),25 (link).
Jumpstart redtaq dna polymerase
Manufactured by Merck Group
Sourced in United States
JumpStart REDTaq DNA polymerase is a thermostable DNA polymerase used for PCR amplification of DNA. It has a 5' to 3' polymerase activity and 3' to 5' exonuclease activity.
Automatically generated - may contain errors
Lab products found in correlation
Cfx96 real time system, by Bio-Rad (3 mentions)
Ez dna methylation kit, by Zymo Research (3 mentions)
Gel extraction kit, by Qiagen (2 mentions)
Topo ta vector system, by Thermo Fisher Scientific (2 mentions)
Nucleospin plasmid isolation kit, by Macherey-Nagel (2 mentions)
Dna methylation kit, by Zymo Research (1 mentions)
Pyromark gold q96 reagent, by Qiagen (1 mentions)
Pyromark q96 id instrument, by Qiagen (1 mentions)
Pyromark q96, by Qiagen (1 mentions)
Pyromark q96 vacuum workstation, by Qiagen (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Picogreen dsdna assay, by Thermo Fisher Scientific (1 mentions)
Longamp taq dna polymerase, by New England Biolabs (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
6 protocols using jumpstart redtaq dna polymerase
1
Quantitative Methylation-Specific PCR Analysis
2
PRF1 Enhancer CpG Methylation Analysis
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DNA amplification of the region containing PRF1 reporter CpG site [–1053 base pairs (bp) upstream of the transcription start site (TSS)] in the enhancer was performed with PCR using Jumpstart REDTaq DNA polymerase (Sigma Aldrich) in T100 Thermal Cycler (Bio‐Rad). One of the primers used for amplification was biotinylated (Biomers) (Supporting information, Table S2 ). Biotinylated PCR amplicons were purified using the Pyromark Q96 Vacuum workstation (Qiagen), followed by pyrosequencing using the Pyromark Q96 ID instrument (Qiagen) with Pyromark Gold Q96 reagent (Qiagen), according to the manufacturer’s protocol. Analysis of the data was performed using Pyromark Q96 software (Qiagen) to determine the CpG methylation profile.
3
Methylation Analysis of miR-5088-5p Promoter
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After genomic DNA was extracted according to a standard phenol-chloroform extraction method, bisulfite modification of genomic DNA was performed using an EZ DNA methylation kit (Zymo Research, USA). The methylation analysis of the miR-5088-5p promoters was performed using MSP primer pairs covering the putative transcriptional start site in the 5′ CpG islands with 1 μℓ of bisulfite-treated DNA as template and JumpStart Red Taq DNA Polymerase (Sigma-Aldrich Company, MO, USA) for amplification as previously described [27 (link)]. DKO (DNMT1(−/−), DNMT3B (−/−) double knockout in HCT116 cells) as unmethylation control, IVD (in vitro methylated DNA) as methylation control, and ddH2O as PCR negative control were used. Primers of the miR-5088-5p promoter region across the upstream from −225 to 34 from mature form sites (Supplementary Fig. S3A ). The sequences of the unmethylation and methylation miR-5088-5p promoter primers used for MSP and qMSP are listed in Table 2 . MSP amplification was performed on bisulfite treated samples and normalized using the Alu element. Real-time PCR was performed by a CFX96TM real-time system (Bio-Rad, Hercules, CA, USA). Alu primer sequence information is previously described [28 (link)].
4
Assessing Genomic Integrity via HPRT Amplification
5
DNA Methylation Analysis by Bisulfite Sequencing
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For methylation analysis, DNA was extracted using a standard phenol-chloroform method. Two micrograms of DNA was modified by bisulfite with the EZ DNA methylation kit™ (Zymo Research, Orange, CA, USA), which guarantees a > 99% conversion rate (non-methylated cytosine to uracil; protection of methylated cytosine residues). Promoter methylation was analyzed using MSP primer pairs located close to the putative transcription start site in the 5′ CpG island, 2 μl of bisulfite-treated DNA as the template, and JumpStart REDTaq DNA polymerase (Sigma-Aldrich), as previously described [18] (link). For bisulfite sequence analysis, PCR amplicons were separated by 2% agarose gel electrophoresis, purified with a gel extraction kit (Qiagen GmbH, Hilden, Germany), and cloned using the TOPO TA vector system (Invitrogen, Carlsbad, CA, USA). Individual clones were isolated and purified using the NucleoSpin plasmid isolation kit (Macherey-Nagel, Düren, Germany). Randomly selected positive clones (10–15 from each sample) were sequenced using the M13F primer, and the methylation status of each CpG dinucleotide was analyzed. For quantification of IL-6 methylation, bisulfite-treated samples were subjected to qMSP. The methylation levels were normalized based on Alu element amplification. qPCR was performed using a CFX96TM real-time system (Bio-Rad, Hercules, CA, USA).
6
Methylation-Specific PCR Analysis of Stem Cell Markers
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For methylation-specific PCR (MSP) analysis, DNA was extracted using the standard phenol-chloroform extraction method. The bisulfite modification of genomic DNA was performed using the EZ DNA methylation kit (Zymo Research, Irvine, CA, USA). We performed the methylation analysis of the CD24, CD44, CD133, and CD147 promoters using MSP primer pairs covering the putative transcriptional start site in the 5′ CpG island with 1 μL of bisulfite-treated DNA template and JumpStart Red Taq DNA Polymerase (Sigma, St. Louis, MO, USA) for amplification, as previously described [32 (link)].
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