Dna methylation kit

Genomic DNA was extracted from the leukocytes in the peripheral blood using an DNA Methylation kit (Zymo, Irvine, CA), following the protocol (Genesky Biotechnologies Inc., Shanghai, China). Genome-wide DNA methylation was assessed using Infinium Human Methylation 850K BeadChip (Illumina Inc., San Diego, CA, United States)). More than 853,000 CpG sites were included in each chip. CpG sites containing documented single-nucleotide polymorphisms and mapping to X and Y chromosomes were removed to avoid potential confounding by single-nucleotide polymorphisms and gender.

Genomic DNA was extracted from ovary, ovotestis and testis tissues, and at least 15 individuals were used in each group using the TIANamp Genomic DNA Kit (Tiangen, China). Concentration and integrity were identified by an Agilent 2100 Bioanalyzer (Agilent, USA) and agarose gel electrophoresis, respectively. Equal amounts of DNA were mixed in the same group and treated using a DNA methylation kit (Zymo, USA) following the manufacturer's protocol. PCR amplification was conducted using treated DNA as the template, and primers were designed by online MethPrimer design software (http://www.urogene.org/methprimer/). The PCR products were purified and cloned into the PMD-18 T vector. A total of 15–20 positive clones from each group were sequenced, and the methylation level was analysed using the DNA methylation analysis platform (http://services.ibc.uni-stuttgart.de/BDPC/BISMA/).

After a medical examination, fasting blood samples were collected from 39 individuals by venipuncture using vacuum anticoagulant tubes with 2 ml EDTA, followed by centrifugation at 3,000 rpm for 10 min to separate plasma. Then 39 samples were stored at −80°C until mtDNA methylation analysis for platelets.
First, 300 μl of plasma was removed from each sample, and platelet mitochondria were extracted using a DNA methylation kit (Zymo Research, Orange, CA, USA) and treated with bisulfite. A 24-μl system was prepared from the bisulfite-treated DNA, and the DNA was amplified by polymerase chain reaction (PCR). Then the DNA was amplified 45 times under the conditions of 5 min at 95°C, 15 s at 95°C, 30 s at 52°C, 15 s at 72°C, and 5 min at 72°C. The PCR amplifications were conducted in a PCR amplifier (BIO-RAD, USA). The treated samples were run on 0.8% agarose gel and visualized under UV light. Finally, the rate of mtDNA methylation was tested by the PyroMark Q48 autoprep pyrophosphate sequencer (QIAGEN, Hilden, Germany) (26 (link)).

TIANamp Blood DNA kit (Tiangen),DNA isolation kit (Qiagen AB, Solna, Sweden), DNA Methylation Kit™ (Zymo Research, HiSS Diagnostics, CA, USA), ZymoTaq™ Pre mix (Zymo Research, HiSS Diagnostics, CA, USA), PyroMark® Q24 pyrosequencer (Qiagen, Germany), RNeasy Plus Mini kit (QIAGEN), ReverTra Ace qPCR RT kit (Toyobo), SYBR-Green PCR master mix (Toyobo).

By methylation-specific polymerase chain reaction (methylation-specific polymerase chain reaction, MSP) method, collection of esophageal squamous cell carcinoma patients who resection of carcinoma tissue and paired normal tissue adjacent to carcinoma, using tissue genomic DNA extraction kit (QIAGEN GmbH) to extract DNA, the DNA methylation kit (ZYMO RESEARCH) for DNA denaturation and bisulfite conversion. The transformed DNA was used as a template for PCR amplification. PRDM5 methylation-specific primers and non-methylation-specific primers (Servicebio) were designed. The methylation-specific primers were upstream 5-TTGTTTCGGGTTTCGCGTTC-3 and downstream 5′-ATTCCTACTACGAAAACGCG-3′. PRDM5 geneno-methylation-specific primers were upstream 5′-TAGTTTTGTTTTGGGTTTTGT-3′ and downstream 5′-CCATTCCTACTACAAAAA CACA-3′. PCR reaction system: DNA template 1 L, 5 × PCR buffer 10 L, upstream and downstream primers 0.5 L each, with ribozyme free water added up to 20 L. PCR reaction conditions: pre-denaturation at 95 °C for 120 s, denaturation at 95 °C for 60s, annealing at 55 °C for 60s, elongation at 72 °C for 60s, 40 cycles. PCR products were analyzed by the gelatinization imaging system. For example, the target band amplified by gene methylation-specific primers indicated positive methylation of the PRDM5 gene, and the gene methylation rate was calculated.

Methylation of the CGIs in the DEP of the SHANK3 promoter region in cINs was evaluated using pyrosequencing. In brief, 0.5 µg of genomic DNA from cINs was bisulfite‐treated using the DNA methylation kit (Zymo Research, Orange, CA, USA). Primers (blue‐highlighted sequences in Figure S2a, Supporting Information) were designed to target the CG loci surrounding the candidate DEP in the SHANK3 promoter region. Bisulfite‐treated DNA was subjected to PCR amplification using the PyroMark PCR Kit (Qiagen) according to the manufacturer's protocol. The PCR amplification conditions were as follows: 95 °C for 10 min, followed by 45 cycles at 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s, and a final step of 72 °C for 10 min. After validation by agarose gel electrophoresis, the PCR product was subjected to quantitative pyrosequencing using PyroMark Q24 (Qiagen). Fully methylated and unmethylated human DNA samples (Zymo Research) were mixed to obtain standard curves. PyroMark Q24 software (Qiagen) was used to quantify the methylation level according to the following formula: methylation(%) = mC/(mC + C), where mC represented methylated cytosine levels and C represented unmethylated cytosine levels.

Genomic DNA from NIH3T3 cells, mESCs and fused cells were extracted using the Genomic DNA Kit (TIANGEN). Bisulphite treatment was performed using the DNA Methylation Kit (ZYMO Research). Then the treated DNA were amplified by touchdown PCR. The primers for OCT4 promoter were 5’- TGG GTT TAT TTA TAT TTA GGA TTT TAGA -3’ and 5’- TCT AAA ACC AAA TAT CCA ACC ATAA -3’ (from −483 to −3), and the primers of Nanog promoter were 5’- TAG GAT ATA GGT TTT TTT TTT AGA TTTG -3’ and 5’- AAC ACC AA C CAA ATC AAC CTATC -3’ (from -717 to-187). Touchdown PCR protocol consisted of two phases. In phase 1, PCR was started with initial denaturation at 98°C for 4 min, 20 cycles of denaturation at 94°C for 45 s, annealing at variable temperatures for 45 s, and extension at 72°C for 1 min. The annealing temperature was set at 66°C in the first cycle and, at each of the 19 subsequent cycles, it was decreased by 0.5°C per cycle down to 56°C. Phase 2 consisted of 20 cycles of 94°C for 45 s, 56°C for 45 s, and 72°C for 1 min. Final step was extension of 8 min at 72°C. Then PCR products were cloned into pUcm-T Vector with pUCm-T Vector Cloning Kit (Sangon Biotech) and individually sequenced as previously described [30 (link)].