Expanded analyses

Using the same BstU I-digested genomic DNA samples, qPCR-based analyses have been tested for several other genes, including leptin (LEP) and tumor necrosis factor alpha (TNF). Except for the primers (designed to have similar melting temperatures, with variation <3°C in any direction), qPCR procedures are identical to the ones used for MDR1. While the results are still considered preliminary and require further confirmation (e.g. by pyrose-quencing and MS-PCR), an important conclusion can be drawn about the comparison of various SYBR® Green qPCR kits: regardless of the gene fragments of interest, the kits from Qiagen consistently produce reliable (clean) amplicons without obvious primer-dimers. Similar kits from three other suppliers either have reduced sensitivity (lower yield) or produce nonspecific amplicons (judged by amplicon size alone) clearly visible on agarose gels stained with ethidium bromide.

For analyses of the MDR1 locus alone, experiments have been further tested in EcoR I-digested gDNA samples treated with other methylation-sensitive DNA endonucleases, including Aci I (CCGC), Hpall (CCGG), Taq I (ACGT), and Xho I (CTCGAG). Other sequences containing any of these sites are also suitable for qPCR-based screening.

Overall, rapid and cost-effective qPCR assays as tested here have at least two major advantages for identifying sequences with differential CpG methylation. First, the tests are quantitative, with highly reproducible results. Second, compared with bisufite-treated, single-stranded DNA samples required for other assays (e.g. MSP and pyrosequencing), the DNA templates treated with methylation-sensitive endonucleases remain double-stranded and can be stored at 4°C for extended and repeated tests. For most conclusive analyses, we recommend qPCR-based screening followed by confirmation with pyrosequencing.

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