DNA was extracted from FLS with the Wizard DNA Clean-Up System (Promega, Madison, WI, United States) according to the manufacturer’s instructions. Unmethylated cytosine residues were converted to uracil with the Methylamp DNA Modification Kit (EpiGentek, Farmingdale, NY, United States). The primer sequences for amplification of methylated and unmethylated PTEN are shown in Supplementary Table S2 .
Methylamp dna modification kit
Manufactured by Epigentek
Sourced in United States
The Methylamp DNA Modification Kit is a laboratory tool designed for the detection and analysis of DNA methylation. The kit provides a standardized method for converting unmethylated cytosine residues in DNA samples to uracil, while leaving methylated cytosines unchanged. This process allows for the study of DNA methylation patterns, a crucial epigenetic mechanism involved in gene regulation.
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Lab products found in correlation
Wizard dna clean up system, by Promega (3 mentions)
Wizard sv gel and pcr clean up system, by Promega (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Pgem t easy vector, by Promega (2 mentions)
Epitect msp kit, by Qiagen (2 mentions)
Tianamp micro dna kit, by Tiangen Biotech (1 mentions)
Pdm19 t vector, by Takara Bio (1 mentions)
Faststart taq dna polymerase, by Merck Group (1 mentions)
Peasy t5 zero cloning kit, by Transgene (1 mentions)
Genomic dna extraction kit, by Tiangen Biotech (1 mentions)
3730xl system, by Thermo Fisher Scientific (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Ez dna methylation direct kit, by Zymo Research (1 mentions)
Sssi methyltransferase, by New England Biolabs (1 mentions)
Gotaq hot start polymerase, by Promega (1 mentions)
Massarray platform, by CapitalBio (1 mentions)
Massarray epityper assay, by Labcorp (1 mentions)
Exosap it, by Thermo Fisher Scientific (1 mentions)
Pcr2.1 vector, by Thermo Fisher Scientific (1 mentions)
Genomic dna purification kit, by Qiagen (1 mentions)
33 protocols using methylamp dna modification kit
1
PTEN Methylation Analysis Protocol
2
Neuronal DNA Methylation Analysis
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DNA was extracted from neuronal cells using a TIANamp Micro DNA Kit (Tiangen, Beijing, China), according to the manufacturer's instructions. DNA was then treated with sodium bisulfite supplied with a Methylamp™ DNA modification kit (Epigentek, Los Angeles, CA, USA), according to the manufacturer's instructions. The bisulfite‐treated DNA was amplified by nested (semi‐nested) PCR for ASIC2a and ASIC4 genes using the primers provided in Table 2 . The PCR products were separated by electrophoresis in 1% agarose gel. The correct bands were excised from the gel and purified using a Wizard® SV Gel and PCR Clean‐Up System (Promega, Madison, WI, USA). The purified DNA was then cloned into a pDM19‐T Vector (TaKaRa), according to the manufacturer's instructions. The positive clones were obtained by antibiotic selection, and the insert was sequenced by Invitrogen (Shanghai, China).
3
Bisulfite Conversion and Amplification of Genomic DNA
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0.5–1 µg of genomic DNA was bisulfite-converted with the Methylamp DNA Modification kit (EPIGENTEK, NY, USA). After the conversion, DNA was amplified using FastStart Taq DNA Polymerase, dNTPack (04738314001, MERCK, Darmstadt, Germany) with the following program: 95°C for 5 min; 4 cycles of 95°C for 1 min, 53°C for 3 min, 72°C for 3 min; 2 cycles of 95°C for 30 s, 55°C for 45 s, 72°C for 45 s; 40 cycles of 95°C for 30 s, 72°C for 1.5 min; 72°C for 10 min. PCR products were purified by QIAquick PCR purification kit (28104, QIAGEN, MD, USA). Further amplicon processing, sequencing, and analysis were performed as described (Sagie et al., 2017b (link)). Primers for amplification of bisulfide converted DNA are described in Supplementary file 1 . In the case in which a primer set amplified several subtelomeres, we used sequence differences between the amplified subtelomeres to identify the origin of the PCR product (Supplementary file 1 , footnote).
4
Promoter Methylation Analysis of Pluripotency Genes
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Genomic DNA was extracted using a genomic DNA extraction kit (TIANGEN, Beijing, China) and the standard protocol. The extracted DNA was then treated with a Methylamp DNA Modification kit (Epigentek, New York, NY, USA) and used as
a template to amplify sequences in the promoter regions of Oct4 and Nanog using a two-round, nested PCR. Subsequent PCR products were cloned into vectors using a pEASY-T5 Zero cloning kit
(TransGen Biotech, Beijing, China). For validation, five randomly selected clones were sequenced.
a template to amplify sequences in the promoter regions of Oct4 and Nanog using a two-round, nested PCR. Subsequent PCR products were cloned into vectors using a pEASY-T5 Zero cloning kit
(TransGen Biotech, Beijing, China). For validation, five randomly selected clones were sequenced.
5
Methylation Analysis of KLF4 in OSCC
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Genomic DNA was extracted from OSCC cell lines using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA), according to the manufacturer's instructions. Genomic DNA was bisulfite-modified with the EZ DNA Methylation-Direct Kit (Zymo Research Co., Orange, CA), and the modified DNA was amplified and examined by electrophoresis in a 1.5% agarose gel to confirm that a single band had been obtained. It was then sequenced with the 3730 xl system (Applied Biosystems, Warrington, United Kingdom). The bisulfite sequencing results were analyzed with BISMA, an online software tool.
The methylation status of KLF4 was further validated in healthy oral mucosa, hyperplasia, dysplasia and OSCC samples. Bisulfite conversion was carried out using the Methylamp DNA Modification Kit (Epigentek, Farmingdale, NY). Methylation analysis was performed using a fluorescence-based, real-time PCR assay with the following conditions: 95°C for 10 min, 35 cycles (95°C 15 s, 60°C 60 s), then 72°C for 7 min.
The methylation status of KLF4 was further validated in healthy oral mucosa, hyperplasia, dysplasia and OSCC samples. Bisulfite conversion was carried out using the Methylamp DNA Modification Kit (Epigentek, Farmingdale, NY). Methylation analysis was performed using a fluorescence-based, real-time PCR assay with the following conditions: 95°C for 10 min, 35 cycles (95°C 15 s, 60°C 60 s), then 72°C for 7 min.
6
MGMT Methylation Analysis by MS-PCR
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After DNA modification with Methylamp DNA Modification Kit (Epigentek, Farmingdale, NY, USA), MS-PCR for MGMT were determined as described previously (Martini et al, 2008 (link)). Briefly, bisulphite-modified DNA (100–200 ng) was amplified in a mixture containing 1 × PCR buffer (20 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2), deoxynucleotide triphosphates (0.2 mM each), primers (20 pM each) and 0.75 U GoTaq Hot Start polymerase (Promega, Madison, WI, USA) in a final volume of 25 μl. Polymerase chain reaction conditions were: an initial denaturation of 95 °C for 8 min, followed by 35 cycles of 95 °C for 60 s, 60 °C for 60 s and 72 °C for 60 s. Polymerase chain reaction products were electrophoresed in a 2.5% agarose gel, stained with ethidium bromide and visualised under ultraviolet illumination. Methylation-specific-PCR analysis was performed in duplicate for all samples. Normal lymphocyte DNA supermethylated with SssI methyltransferase (New England Biolabs, Beverly, MA, USA) and treated with bisulphite was used as the unmethylated and methylated control, water as a negative control and untreated DNA as an internal PCR control. We also carried out MS-PCR on granulocyte DNA obtained from 10 healthy individuals, as the control group.
7
Bisulfite Sequencing Analysis of DNA Methylation
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A total of 200 ng of genomic DNA from each sample was bisulfite-treated with the Methylamp DNA Modification Kit (Epigentek). The quality of the bisulfite conversion was controlled using PCR products that had no methyl group. The Sequenom MASSARRAY platform (CapitalBio, Beijing, China) uses matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry in combination with RNA base-specific cleavage (MassCLEAVE). A detectable pattern is then analyzed for its methylation status [15] (link). PCR primers were designed with Methprimer (http://epidesigner.com ), and the primer sequence can be found in Table S1 .
8
MassARRAY EpiTYPER Assay for DMR Validation
9
Bisulfite Sequencing and HUMARA Assay for ATP6AP1 Methylation
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For bisulfite sequencing54 (link) of ATP6AP1 mutations near CpG islands, 200 ng of tumor DNA was treated with a bisulfite conversion kit (Methylamp DNA Modification Kit; EpiGentek), and amplified by PCR with methylated and non-methylated DNA-specific primers. For the modified HUMARA assay55 (link) following methylation-specific DNA digestion56 (link), 200 ng of tumor DNA was incubated overnight at 37 °C with 25 U of the methylation-sensitive restriction enzyme HhaI (New England Biolabs) or water (for mock-digested control). PCR fragments were cleaned using ExoSAP-IT (ThermoFisher Scientific) and subjected to Sanger sequencing as previously described23 (link).
10
Imprinted Gene Methylation Analysis
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Genomic DNA was extracted using a genomic DNA purification kit (QIAGEN). Two hundred nanograms of genomic DNA from each sample was treated with a Methylamp DNA modification kit (P-1001-1, Epigentek, Brooklyn, NY, USA) to convert the unmethylated cytosine to uracil according to the manufacturer’s instructions. The three imprinted genes H19, HEG1, and SNRPN amplified by PCR using EX Taq HS (Biomed), cloned into the PCR 2.1 vector (Invitrogen), and sequenced with M13. PCR primers were summarized in Additional file 2 : Table S2.
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