Epitect bisul te kit

Manufactured by Qiagen

Sourced in Germany

The EpiTect Bisulfite Kit is a product designed for the conversion of unmethylated cytosine to uracil in DNA samples, a crucial step in the process of DNA methylation analysis. The kit provides reagents and protocols to perform this bisulfite conversion efficiently and reliably.

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21 protocols using epitect bisul te kit

Methylation analysis of BNIP3 in GBC

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Extraction of Genomic DNA from gallstone and GBC tissue samples were performed by standard phenol/chloroform extraction protocol. EpiTect Bisul te Kit (Qiagen GmbH, Germany) was used for Sodium bisul te modi cation of extracted DNA followed by manufacturer's instructions. MSP was performed using DreamTaq TM Hot Start PCR Master Mix (Thermo Scienti c, US) and primer sets speci c for methylated or unmethylated sequences for promoter region of BNIP3 (Supplementary Table 1).
Bisul te converted lymphocyte DNA was used as positive control for unmethylated primer while same DNA treated with SssI methyltransferase (NEB, Beverly, MA) as positive control for methylated primer and water blank as negative control for each set of primers. Methylation-speci c PCR was repeated in duplicate. To validate the MSP results and to con rm the methylation status, Sanger sequencing was done in one heterozygous methylated GBC sample and one unmethylated gallstone sample (Supplementary gure 1).

Bharti A., Kar A.G., Singh D., Ansari M.A., Tewari M., Narayan G, & Singh S. (2022). Frequent promoter hypermethylation and down regulation of BNIP3: An early event during gallbladder cancer progression. Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver, 54(9).

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DNA Extraction and Bisulfite Modification

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DNA was extracted from peripheral blood samples using a commercial DNA extraction kit (QIAamp DNA Blood Mini Kit, Hilden, Germany). The concentration and the purity of DNA were detected by Nanodrop 2000 Spectrophotometer (Thermo Scienti c). Genomic DNA was bisul te-modi ed with an EpiTect Bisul te Kit (Qiagen, Hilden, Germany). Bisul te DNA was normalized at a concentration of 20 ng/mL and was stored at - 20 °C for the following experiment. DNA extraction and DNA sodium bisul te modi cation were performed according to the manufacturer's instructions.

Ge A., Gao S., Liu Y., Zhang H., Wang X., Zhang L., Pang D., & Zhao Y. (2020). Joint effects of WT1, CA10 methylation in peripheral blood leukocyte and dietary zinc on breast cancer risk: a case-control study.

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Promoter Methylation Analysis of NKILA

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Sodium bisul te conversion was conducted with EpiTect Bisul te Kit (Qiagen). Afterwards, MSP was performed in bisul te-treated DNA with two sets of primers, which were speci c to unmethylated (U-MSP) or methylated (M-MSP) DNA sequence. MSP primers were designed at the CpG island upstream to NKILA gene by online tool Methprimer (http://www.urogene.org/methprimer/). Details of primer sequence and PCR condition for MSP were listed in Table 1. For each MSP reaction, the enzymatically methylated control DNA (CpGenome Universal Methylated DNA; Chemicon / Millipore, Billerica, MA, USA) was used as positive control for M-MSP and negative control for U-MSP. Total RNA was isolated with the mirVanaTM miRNA Isolation Kit (Invitrogen), followed by reverse transcription with SuperScript® III (Invitrogen). qRT-PCR was performed with SYBR® Select Master Mix (ABI), and the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control. The relative quantity of NKILA expression was calculated by the method of 2-ΔΔCt and normalized against the endogenous control. The primer sequences for NKILA and GAPDH were listed in Table 1 [19, 21] .

Zhang M.Y., Deng M.D., Au-Yeung R., Wang L.Q., & Chim C.S. (2020). Epigenetic Silencing of Tumor Suppressor IncRNA NKILA: Implication on NF-κB Signaling in Non-Hodgkin’s Lymphoma.

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Methylation Analysis of ITPR3 Promoter

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CpG islands are closely related to the methylation level of genes. CpG islands in the promoter region of ITPR3 were predicted by MethPrimer (http://www.urogene.org/methprimer/). Genomic DNA (1 µg) from bladder cancer cells and SV-HUC-1 cells was modi ed and puri ed with sodium bisul te using the EpiTect Bisul te Kit (Qiagen, cat: 59824 Hilden, Germany). Bisul te genomic sequencing was used to analyze the methylated CpGs in the ITPR3 promoter, and the primers used for bisul te sequencing PCR were ITPR3 F: 5'-ATTTGTATGTGTGTGGTGGTTT-3' (sense) and 5'-TAAAACCATTAACRAAACCCTC-3' (antisense). Ampli ed bisulfate PCR products were cloned into the pMD18-T simple vector (Takara, Dalian, China). Ten bacterial colonies containing 43 methylation sites were selected for sequencing at Sangon Biotech (Shanghai, China).

Zhang M., Wang L., Yue Y., Zhang L., Liu T., Jing M., Zhang Y., Ma M., Xu S., Wang K., Wang X., & Fan J. (2021). ITPR3 facilitates tumor growth, metastasis and stemness by inducing the NF-ĸB/CD44 pathway in urinary bladder carcinoma.

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DNA Extraction and Bisulfite Sequencing

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Genomic DNA extraction and bisul te conversion were performed as described previously. [22] Brie y, genomic DNA was puri ed using a Gentra Puregene Cell Kit (Qiagen). Bisul te conversion was performed using an EpiTect Bisul te Kit (Qiagen). Bisul ted DNA was ampli ed with Takara Epi-Taq HS (Takara Bio) using a speci c primer for the human UQCRH CpG island shore. The primer sequences are listed in Table 5. After ligation with the pCR4-TOPO vector using the TOPO TA Cloning Kit (Thermo Fisher Scienti c), products were transformed into DH5a and sequenced.

Miyakuni K., Nishida J., Koinuma D., Nagae G., Aburatani H., Miyazono K., & Ehata S. (2021). Genome-wide analysis of DNA methylation identifies the apoptosis-related gene UQCRH in renal cancer.

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Molecular Mechanisms of miR-133a in Glioma Regulation

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The human normal glial cell line HEB and glioma cell lines U251, U87, T98-G and A172 were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DMEM/HG, fetal bovine serum (FBS), Opti-MEM and 0.25% trypsin containing 0.02% EDTA were purchased from Gibco, MTT kit, 5-aza-2'-deoxycytosine (AZA) and histone deacetylase inhibitor (TSA) from Sigma, Trizol, reverse transcription kit from Thermo, SYBR Green Real-time PCR kit from Shanghai Solarbio Bioscience & Technology, QIAamp DNA Mini kit and EpiTect Bisul te kit from Qiagen, Lipofectamine 3000 from Invitrogen, and the cell cycle assay kit from BD. The pcDNA3.1-miR-133a mimic, scrambled miRNA (miR-NC) and the pcDNA3.1-PPARγ overexpression plasmid were obtained from Guangzhou Ruibo Bio. Antibodies against PPARγ, cyclin D1, cyclin D2, CDK4 and β-actin were from Abcam, and the horseradish peroxidase (HRP)-labeled IgG secondary antibody from Guangzhou Jingcai. PPARγ wild type (WT) and mutant (MUT) luciferase reporter plasmids were purchased from Shanghai Jima and the luciferase assay kit from Promega. The PCR primers were synthesized by Shanghai Shenggong. Other reagents were from our laboratory and of analytical grade.

Liu L., Zhu Z., Li X., & Zheng Y. (2020). DNA methylation regulates glioma cell cycle through down-regulating MiR-133a expression.

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DNA Extraction and Bisulfite Modification

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DNA was extracted from peripheral blood samples using a commercial DNA extraction kit (QIAamp DNA Blood Mini Kit, Hilden, Germany). The concentration and the purity of DNA were assessed using a Nanodrop 2000 Spectrophotometer (Thermo Scienti c, USA). Genomic DNA was bisul te-modi ed with an EpiTect Bisul te kit (Qiagen, Hilden, Germany).
Bisul te DNA was normalized to a concentration of 20 ng/mL and was stored at -20°C for the following experiment. DNA extraction and DNA sodium bisul te modi cation were performed according to the manufacturers' instructions.

Ge A., Gao S., Liu Y., Zhang H., Wang X., Zhang L., Pang D., & Zhao Y. (2020). Methylation of WT1, CA10 in peripheral blood leukocyte is associated with breast cancer risk: a case-control study.

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Methylation Analysis of ITPR3 Promoter

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Zhang M., Wang L., Yue Y., Zhang L., Liu T., Jing M., Liang X., Ma M., Xu S., Wang K., Wang X., & Fan J. (2020). ITPR3 Facilitates Tumor Growth, Metastasis and Stemness by Inducing the NF-ĸB/CD44 Pathway in Urinary Bladder Carcinoma.

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Illumina EPIC Methylation Profiling

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Illumina EPIC chip processing Genomic DNA was extracted using standard protocols 21, 22 . Whole-blood genomic DNA diluted with water (50 ng/µl) was treated with sodium bisul te using the EpiTect® Bisul te Kit from QIAGEN® following the manufacturer's protocol. DNAm was assessed using the Illumina In nium HumanMethylationEPIC BeadChip kit (Illumina, Inc., San Diego, California) and quanti ed on an Illumina HiScan System (Illumina, Inc.). The level of methylation is expressed as a 'beta' value (β-value), ranging from 0 (no cytosine methylation) to 1 (complete cytosine methylation). 23 . Data pre-processing and downstream statistical analyses were conducted using R version 3.6.0 24 .

Alameda L., Liu Z., Sham P.C., Aas M., Trotta G., Rodriguez V., Di Forti M., Stilo S.A., Kandaswamy R., Arango C., Arrojo M., Bernardo M., Bobes J., de Haan L., Del-Ben C.M., Gayer-Anderson C., Sideli L., Jones P.B., Jongsma H.E., Kirkbride J.B., La Cascia C., Lasalvia A., Tosato S., Llorca P.M., Menezes P.R., van Os J., Quattrone D., Rutten B.P., Santos J.L., Sanjuán J., Selten J.P., Szöke A., Tarricone I., Tortelli A., Velthorst E., Morgan C., Dempster E., Hannon E., Burrage J., Dwir D., Arumuham A., Mill J., Murray R.M, & Wong C.C.Y. (2023). Exploring the mediation of DNA methylation across the epigenome between childhood adversity and First Episode of Psychosis-findings from the EU-GEI study. Molecular psychiatry, 28(5).

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ZIC4 Promoter Methylation Analysis

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DNA was isolated using the proteinase K/phenol extraction method. Bisul te conversion was carried out using 1 µg of DNA using an Epitect Bisul te Kit (Qiagen). Bisul te-treated DNA was ampli ed with BSP primers located in the ZIC4 promoter and PCR products were cloned using the pGEM-T Easy Vector system (Promega, Madison, WI). Three individual clones were sequenced. The region assessed by BSP included 8 CpG sites from the ZIC4 promoter and average methylation from individual clones was calculated as a percentage of the number of methylated CpG sites over the number of total CpG sites sequenced.

Wang J., Yang Z., Long F., Luo L., Gao L., Chen C., Sun D., & Tang D. (2020). EZH2-mediated epigenetic silencing of ZIC4 in hepatocellular carcinoma.

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