EpiTect Bisulfite Kit

DNA methylation profiling Gene specific profiling - HEK293T RHO

Experiment
DNA methylation profiling Gene specific profiling - HEK293T RHO
Product
EpiTect Bisulfite Kit from Qiagen
Manufacturer
Qiagen

Protocol tips

Protocol tips
For best results, the CT Conversion Reagent should be prepared freshly and used immediately following preparation.

Publication protocol

Bisulfite Sequencing
Bisulfite modification of genomic DNA was performed using either the Epitech (Qiagen, Valencia, CA) or EZ DNA Methylation (Zymo, Irvine, CA) kits. MethPrimer [46] was used to design primers to specifically amplify photoreceptor specific genes and the putative promoter regions of two intergenic miRNAs (Mir183 and Mir184) (Tables S2, S3, S4). For direct sequencing, the PCR products were agarose gel-purified and directly sequence by the High-Throughput Genomics Unit at the University of Washington using the forward primers. Using the ContigExpress function of Vector NTI Advance 10 (Invitrogen, Carlsbad, CA), the chromatograph peak heights for C and T at each CpG site were measured and the % methylation (C peak height/(T peak height+C peak height)) versus CpG position, with respect to the TSS, was plotted [47]. For sequencing of individual clones after PCR amplification, the presence of a specific PCR product was confirmed by agarose gel electrophoresis, and the PCR product for each set of primers was subcloned using the TOPO-TA cloning kit (Invitrogen, Valencia, CA). Thirty sequences (10 clones for each of 3 biological replicates) with ≥95% conversion of the non-CpG cytosines were obtained and sequenced using the M13 reverse primer by the University of Washington High-throughput Genomics Unit. Fasta files of each sequence were analyzed using a custom JAVA program (DNAMethylMap, unpublished software by B. Kumar and S. Yegnasubramanian).

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Discussion

Discussion

2 years ago

Author: Jody Hancock Canada

How can I preserve leukocytes for DNA methylation profiling?

I would like to preserve leukocytes for future epigenetic analysis. How can I preserve them effectively in order to perform DNA methylation profiling at a later time?

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Papers

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Manufacturer protocol

Download the product protocol from Qiagen for EpiTect Bisulfite Kit below.

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