My experimental gene has 5 different transcription start sites.From different web based tools, I searched that the predominance of these sites in different tissues but still now I have not get any satisfactory answer.Because I am working with cell lines, it might be possible that whatever result, I get in tissues, is not valid for cell line.Generally it is considered that from transcription start site to the upstream 1500bp region, is the promoter site. I want to analyse the promoter of my gene but because of the variation of transcription start sites, I am a little bit confused. Should I do a 5-RACE assay in my cell line?How could I progress further? Any in depth advice is acceptable.Thank you.
If the TSSs are experimentally proven (not only by online analysis tools), then you can take upstream DNA region containing all the TSSs because that constitutes the real promoter region (all the TSSs might affect gene expression at a given time). Otherwise, you may have to use 5' RNA ligase mediated RACE protocol to experimentally validate all the TSSs. Hope the suggestion helps...
With the explosion of large datasets, whole genome transcription analyses, and bioinformatic annotation tools, many of the classic methods for mapping real TSS have been lost. For any given cel line or tissue, it is impossible to know which TSS are being used unless you actually map them. 5' RACE, primer extension, S1 nuclease mapping are all methods that can determine the TSS within a few nucleotides. Alternatively, you can construct expression plasmids using the various potential promoters and see which ones work best in your cell line for expressing a reporter like luciferase.
Dear Gregory,
My gene has internal start site.So, if I will do S1 mapping, it would cut both longer one and the shorter one also because, primer hybridize with both of them.Should I go with RACE?
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